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Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that IL-13-activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibited viscoelastic liquid behavior and that mucus secreted by IL-13-activated HAECs exhibited solid-like behavior caused by mucin cross-linking. In addition, IL-13-activated HAECs shows increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that was prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), was increased in IL-13-activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings was increased in patients with asthma with high airway mucus plug scores. Together, our results show that IL-13-activated HAECs autonomously generated pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

BACKGROUNDInformation about the size, airway location, and longitudinal behavior of mucus plugs in asthma is needed to understand their role in mechanisms of airflow obstruction and to rationally design muco-active treatments.METHODSCT lung scans from 57 patients with asthma were analyzed to quantify mucus plug size and airway location, and paired CT scans obtained 3 years apart were analyzed to determine plug behavior over time. Radiologist annotations of mucus plugs were incorporated in an image-processing pipeline to generate size and location information that was related to measures of airflow.RESULTSThe length distribution of 778 annotated mucus plugs was multimodal, and a 12 mm length defined short ("stubby", ≤12 mm) and long ("stringy", >12 mm) plug phenotypes. High mucus plug burden was disproportionately attributable to stringy mucus plugs. Mucus plugs localized predominantly to airway generations 6-9, and 47% of plugs in baseline scans persisted in the same airway for 3 years and fluctuated in length and volume. Mucus plugs in larger proximal generations had greater effects on spirometry measures than plugs in smaller distal generations, and a model of airflow that estimates the increased airway resistance attributable to plugs predicted a greater effect for proximal generations and more numerous mucus plugs.CONCLUSIONPersistent mucus plugs in proximal airway generations occur in asthma and demonstrate a stochastic process of formation and resolution over time. Proximal airway mucus plugs are consequential for airflow and are in locations amenable to treatment by inhaled muco-active drugs or bronchoscopy.TRIAL REGISTRATIONClinicaltrials.gov; NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01716494, and NCT01760915.FUNDINGAstraZeneca, Boehringer-Ingelheim, Genentech, GlaxoSmithKline, Sanofi-Genzyme-Regeneron, and TEVA provided financial support for study activities at the Coordinating and Clinical Centers beyond the third year of patient follow-up. These companies had no role in study design or data analysis, and the only restriction on the funds was that they be used to support the SARP initiative.

Abstract: Importance: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or other health effects after the acute phase of infection; termed post-acute sequelae of SARS-CoV-2 infection (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are ill-defined. The objectives of the Researching COVID to Enhance Recovery (RECOVER) Multi-site Observational Study of PASC in Adults (RECOVER-Adult) are to: (1) characterize PASC prevalence; (2) characterize the symptoms, organ dysfunction, natural history, and distinct phenotypes of PASC; (3) identify demographic, social and clinical risk factors for PASC onset and recovery; and (4) define the biological mechanisms underlying PASC pathogenesis. Methods: RECOVER-Adult is a combined prospective/retrospective cohort currently planned to enroll 14,880 adults aged ≥18 years. Eligible participants either must meet WHO criteria for suspected, probable, or confirmed infection; or must have evidence of no prior infection. Recruitment occurs at 86 sites in 33 U.S. states, Washington, DC and Puerto Rico, via facility– and community-based outreach. Participants complete quarterly questionnaires about symptoms, social determinants, vaccination status, and interim SARS-CoV-2 infections. In addition, participants contribute biospecimens and undergo physical and laboratory examinations at approximately 0, 90 and 180 days from infection or negative test date, and yearly thereafter. Some participants undergo additional testing based on specific criteria or random sampling. Patient representatives provide input on all study processes. The primary study outcome is onset of PASC, measured by signs and symptoms. A paradigm for identifying PASC cases will be defined and updated using supervised and unsupervised learning approaches with cross– validation. Logistic regression and proportional hazards regression will be conducted to investigate associations between risk factors, onset, and resolution of PASC symptoms. Discussion: RECOVER-Adult is the first national, prospective, longitudinal cohort of PASC among US adults. Results of this study are intended to inform public health, spur clinical trials, and expand treatment options.

Rationale: The role of obesity-associated insulin resistance (IR) in airflow limitation in asthma is uncertain. Objectives: Using data in the Severe Asthma Research Program 3 (SARP-3), we evaluated relationships between homeostatic measure of IR (HOMA-IR), lung function (cross-sectional and longitudinal analyses), and treatment responses to bronchodilators and corticosteroids. Methods: HOMA-IR values were categorized as without (5.0). Lung function included FEV1 and FVC measured before and after treatment with inhaled albuterol and intramuscular triamcinolone acetonide and yearly for 5 years. Measurements and Main Results: Among 307 participants in SARP-3, 170 (55%) were obese and 140 (46%) had IR. Compared with patients without IR, those with IR had significantly lower values for FEV1 and FVC, and these lower values were not attributable to obesity effects. Compared with patients without IR, those with IR had lower FEV1 responses to β-adrenergic agonists and systemic corticosteroids. The annualized decline in FEV1 was significantly greater in patients with moderate IR (-41 ml/year) and severe IR (-32 ml/year,) than in patients without IR (-13 ml/year, P

BackgroundHeterozygote carriers of potentially pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have increased asthma risk. However, the frequency and impact of CFTR variation among individuals with asthma is unknown.ObjectiveTo determine whether potentially pathogenic CFTR variants associate with disease severity and whether individuals with two potentially pathogenic variants exist in a severe asthma-enriched cohort.MethodsWe analyzed sequencing data spanning a 190.5Kb region of CFTR in participants from the Severe Asthma Research Program (SARP1-3). Potentially pathogenic, rare CFTR variants (frequency  G] and one Arg1070Trp) and a homozygote for the VVCC, 5 T; TG12.ConclusionsWe found potentially pathogenic CFTR variants within a severe asthma-enriched cohort, including three compound heterozygote genotypes variably associated with CF in NHW individuals. These findings provide the rationale for CFTR sequencing and phenotyping of CF-related traits in individuals with severe asthma.

The Collaborative Cohort of Cohorts for COVID-19 Research (C4R) is a national prospective study of adults comprising 14 established US prospective cohort studies. Starting as early as 1971, investigators in the C4R cohort studies have collected data on clinical and subclinical diseases and their risk factors, including behavior, cognition, biomarkers, and social determinants of health. C4R links this pre-coronavirus disease 2019 (COVID-19) phenotyping to information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and acute and postacute COVID-related illness. C4R is largely population-based, has an age range of 18-108 years, and reflects the racial, ethnic, socioeconomic, and geographic diversity of the United States. C4R ascertains SARS-CoV-2 infection and COVID-19 illness using standardized questionnaires, ascertainment of COVID-related hospitalizations and deaths, and a SARS-CoV-2 serosurvey conducted via dried blood spots. Master protocols leverage existing robust retention rates for telephone and in-person examinations and high-quality event surveillance. Extensive prepandemic data minimize referral, survival, and recall bias. Data are harmonized with research-quality phenotyping unmatched by clinical and survey-based studies; these data will be pooled and shared widely to expedite collaboration and scientific findings. This resource will allow evaluation of risk and resilience factors for COVID-19 severity and outcomes, including postacute sequelae, and assessment of the social and behavioral impact of the pandemic on long-term health trajectories.

Rationale: Cross-sectional analysis of mucus plugs in computed tomography (CT) lung scans in the Severe Asthma Research Program (SARP)-3 showed a high mucus plug phenotype. Objectives: To determine if mucus plugs are a persistent asthma phenotype and if changes in mucus plugs over time associate with changes in lung function. Methods: In a longitudinal analysis of baseline and Year 3 CT lung scans in SARP-3 participants, radiologists generated mucus plug scores to assess mucus plug persistence over time. Changes in mucus plug score were analyzed in relation to changes in lung function and CT air trapping measures. Measurements and Main Results: In 164 participants, the mean (range) mucus plug score was similar at baseline and Year 3 (3.4 [0-20] vs. 3.8 [0-20]). Participants and bronchopulmonary segments with a baseline plug were more likely to have plugs at Year 3 than those without baseline plugs (risk ratio, 2.8; 95% confidence interval [CI], 2.0-4.1; P

BackgroundThe large airway epithelial barrier provides one of the first lines of defense against respiratory viruses, including SARS-CoV-2 that causes COVID-19. Substantial inter-individual variability in individual disease courses is hypothesized to be partially mediated by the differential regulation of the genes that interact with the SARS-CoV-2 virus or are involved in the subsequent host response. Here, we comprehensively investigated non-genetic and genetic factors influencing COVID-19-relevant bronchial epithelial gene expression.MethodsWe analyzed RNA-sequencing data from bronchial epithelial brushings obtained from uninfected individuals. We related ACE2 gene expression to host and environmental factors in the SPIROMICS cohort of smokers with and without chronic obstructive pulmonary disease (COPD) and replicated these associations in two asthma cohorts, SARP and MAST. To identify airway biology beyond ACE2 binding that may contribute to increased susceptibility, we used gene set enrichment analyses to determine if gene expression changes indicative of a suppressed airway immune response observed early in SARS-CoV-2 infection are also observed in association with host factors. To identify host genetic variants affecting COVID-19 susceptibility in SPIROMICS, we performed expression quantitative trait (eQTL) mapping and investigated the phenotypic associations of the eQTL variants.ResultsWe found that ACE2 expression was higher in relation to active smoking, obesity, and hypertension that are known risk factors of COVID-19 severity, while an association with interferon-related inflammation was driven by the truncated, non-binding ACE2 isoform. We discovered that expression patterns of a suppressed airway immune response to early SARS-CoV-2 infection, compared to other viruses, are similar to patterns associated with obesity, hypertension, and cardiovascular disease, which may thus contribute to a COVID-19-susceptible airway environment. eQTL mapping identified regulatory variants for genes implicated in COVID-19, some of which had pheWAS evidence for their potential role in respiratory infections.ConclusionsThese data provide evidence that clinically relevant variation in the expression of COVID-19-related genes is associated with host factors, environmental exposures, and likely host genetic variation.

Rationale: Androgens are potentially beneficial in asthma, but AR (androgen receptor) has not been studied in human airways.Objectives: To measure whether AR and its ligands are associated with human asthma outcomes.Methods: We compared the effects of AR expression on lung function, symptom scores, and fractional exhaled nitric oxide (FeNO) in adults enrolled in SARP (Severe Asthma Research Program). The impact of sex and of androgens on asthma outcomes was also evaluated in the SARP with validation studies in the Cleveland Clinic Health System and the NHANES (U.S. National Health and Nutrition Examination Survey).Measurements and Main Results: In SARP (n = 128), AR gene expression from bronchoscopic epithelial brushings was positively associated with both FEV1/FVC ratio (R2 = 0.135, P = 0.0002) and the total Asthma Quality of Life Questionnaire score (R2 = 0.056, P = 0.016) and was negatively associated with FeNO (R2 = 0.178, P = 9.8 × 10-6) and NOS2 (nitric oxide synthase gene) expression (R2 = 0.281, P = 1.2 × 10-10). In SARP (n = 1,659), the Cleveland Clinic Health System (n = 32,527), and the NHANES (n = 2,629), women had more asthma exacerbations and emergency department visits than men. The levels of the AR ligand precursor dehydroepiandrosterone sulfate correlated positively with the FEV1 in both women and men.Conclusions: Higher bronchial AR expression and higher androgen levels are associated with better lung function, fewer symptoms, and a lower FeNO in human asthma. The role of androgens should be considered in asthma management.

Rationale: It is unclear why select patients with moderate-to-severe asthma continue to lose lung function despite therapy. We hypothesized that participants with the smallest responses to parenteral corticosteroids have the greatest risk of undergoing a severe decline in lung function.Objectives: To evaluate corticosteroid-response phenotypes as longitudinal predictors of lung decline.Methods: Adults within the NHLBI SARP III (Severe Asthma Research Program III) who had undergone a course of intramuscular triamcinolone at baseline and at ≥2 annual follow-up visits were evaluated. Longitudinal slopes were calculated for each participant's post-bronchodilator FEV1% predicted. Categories of participant FEV1 slope were defined: severe decline, >2% loss/yr; mild decline, >0.5-2.0% loss/yr; no change, 0.5% loss/yr to

Rationale: Some reports indicate longitudinal variability in sputum differential cell counts, whereas others describe stability. Highly variable sputum eosinophil percentages are associated with greater lung function loss than persistently elevated eosinophil percentages, but elevated neutrophils are linked to more severe asthma.Objectives: To examine sputum granulocyte stability or variability longitudinally and associations with important clinical characteristics.Methods: The SARP III (Severe Asthma Research Program III) cohort underwent comprehensive phenotype characterization at baseline and annually over 3 years. Adult subjects with acceptable sputum levels were assigned to one of three longitudinal sputum groups: eosinophils predominantly 2 SDs determined from independent, repeated baseline eosinophil percentages). Subjects were similarly assigned to one of three longitudinal neutrophil groups with a 50% cut point.Measurements and Main Results: The group with predominantly

BackgroundThe Chr17q12-21.2 region is the strongest and most consistently associated region with asthma susceptibility. The functional genes or single nucleotide polymorphisms (SNPs) are not obvious due to linkage disequilibrium.ObjectivesWe sought to comprehensively investigate whole-genome sequence and RNA sequence from human bronchial epithelial cells to dissect functional genes/SNPs for asthma severity in the Severe Asthma Research Program.MethodsExpression quantitative trait loci analysis (n = 114), correlation analysis (n = 156) of gene expression and asthma phenotypes, and pathway analysis were performed in bronchial epithelial cells and replicated. Genetic association for asthma severity (426 severe vs 531 nonsevere asthma) and longitudinal asthma exacerbations (n = 273) was performed.ResultsMultiple SNPs in gasdermin B (GSDMB) associated with asthma severity (odds ratio, >1.25) and longitudinal asthma exacerbations (P < .05). Expression quantitative trait loci analyses identified multiple SNPs associated with expression levels of post-GPI attachment to proteins 3, GSDMB, or gasdermin A (3.1 × 10-9 -4). Higher expression levels of GSDMB correlated with asthma and greater number of exacerbations (P < .05). Expression levels of GSDMB correlated with genes involved in IFN signaling, MHC class I antigen presentation, and immune system pathways (false-discovery rate-adjusted P < .05). rs1031458 and rs3902920 in GSDMB colocalized with IFN regulatory factor binding sites and associated with GSDMB expression, asthma severity, and asthma exacerbations (P < .05).ConclusionsBy using a unique set of gene expression data from lung cells obtained using bronchoscopy from comprehensively characterized subjects with asthma, we show that SNPs in GSDMB associated with asthma severity, exacerbations, and GSDMB expression levels. Furthermore, its expression levels correlated with asthma exacerbations and antiviral pathways. Thus, GSDMB is a functional gene for both asthma susceptibility and severity.

Rationale: Cross-sectional studies suggest an exacerbation-prone asthma (EPA) phenotype and the utility of blood eosinophils and plasma IL-6 as predictive biomarkers.Objectives: To prospectively test for EPA phenotype and utility of baseline blood measures of eosinophils and IL-6 as predictive biomarkers.Methods: Three-year asthma exacerbation data were analyzed in 406 adults in the Severe Asthma Research Program-3. Transition models were used to assess uninformed and informed probabilities of exacerbation in year 3. Binomial regression models were used to assess eosinophils and IL-6 as predictive biomarkers.Measurements and Main Results: Eighty-three participants (21%) had ≥1 exacerbation in each year (EPA) and 168 participants (41%) had no exacerbation in any year (exacerbation-resistant asthma). The uninformed probability of an exacerbation in Year 3 was 40%, but the informed probability increased to 63% with an exacerbation in Year 2 and 82% with an exacerbation in Years 1 and 2. The probability of a Year 3 exacerbation with no Year 1 or 2 exacerbations was 13%. Compared with exacerbation-resistant asthma, EPA was characterized by lower FEV1 and a higher prevalence of obesity, hypertension, and diabetes. High-plasma IL-6 occurred in EPA, and the incident rate ratio for exacerbation increased 10% for each 1-pg/μl increase in baseline IL-6 level. Although high blood eosinophils did not occur in EPA, the incident rate ratio for exacerbations increased 9% for each 100-cell/μl increase in baseline eosinophil number.Conclusions: Longitudinal analysis confirms an EPA phenotype characterized by features of metabolic dysfunction. Blood measures of IL-6, but not eosinophils, were significantly associated with EPA, and IL-6 and eosinophils predicted exacerbations in the sample as a whole.

Rationale: Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 (angiotensin-converting enzyme 2), and TMPRSS2 (transmembrane protease serine 2) mediate viral infection of host cells. We reasoned that differences in ACE2 or TMPRSS2 gene expression in sputum cells among patients with asthma may identify subgroups at risk for COVID-19 morbidity.Objectives: To determine the relationship between demographic features and sputum ACE2 and TMPRSS2 gene expression in asthma.Methods: We analyzed gene expression for ACE2 and TMPRSS2, and for ICAM-1 (intercellular adhesion molecule 1) (rhinovirus receptor as a comparator) in sputum cells from 330 participants in SARP-3 (Severe Asthma Research Program-3) and 79 healthy control subjects.Measurements and Main Results: Gene expression of ACE2 was lower than TMPRSS2, and expression levels of both genes were similar in asthma and health. Among patients with asthma, male sex, African American race, and history of diabetes mellitus were associated with higher expression of ACE2 and TMPRSS2. Use of inhaled corticosteroids (ICS) was associated with lower expression of ACE2 and TMPRSS2, but treatment with triamcinolone acetonide did not decrease expression of either gene. These findings differed from those for ICAM-1, where gene expression was increased in asthma and less consistent differences were observed related to sex, race, and use of ICS.Conclusions: Higher expression of ACE2 and TMPRSS2 in males, African Americans, and patients with diabetes mellitus provides rationale for monitoring these asthma subgroups for poor COVID-19 outcomes. The lower expression of ACE2 and TMPRSS2 with ICS use warrants prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID-19 morbidity.

Combined elevated sputum eosinophils+neutrophils in asthma associated with lowest lung function, greater healthcare utilization, and longitudinally, further spirometric loss, implicating cell-cell interactions or overlapping inflammatory pathways while increased eosinophils or neutrophils alone show less effect.

BackgroundRelative enlargement of the pulmonary artery (PA) on chest CT imaging is associated with respiratory exacerbations in patients with COPD or cystic fibrosis. We sought to determine whether similar findings were present in patients with asthma and whether these findings were explained by differences in ventricular size.MethodsWe measured the PA and aorta diameters in 233 individuals from the Severe Asthma Research Program III cohort. We also estimated right, left, and total epicardial cardiac ventricular volume indices (eERVVI, eELVVI, and eETVVI, respectively). Associations between the cardiac and PA measures (PA-to-aorta [PA/A] ratio, eERVVI-to-eELVVI [eRV/eLV] ratio, eERVVI, eELVVI, eETVVI) and clinical measures of asthma severity were assessed by Pearson correlation, and associations with asthma severity and exacerbation rate were evaluated by multivariable linear and zero-inflated negative binomial regression.ResultsAsthma severity was associated with smaller ventricular volumes. For example, those with severe asthma had 36.1 mL/m2 smaller eETVVI than healthy control subjects (P = .003) and 14.1 mL/m2 smaller eETVVI than those with mild/moderate disease (P = .011). Smaller ventricular volumes were also associated with a higher rate of asthma exacerbations, both retrospectively and prospectively. For example, those with an eETVVI less than the median had a 57% higher rate of exacerbations during follow-up than those with eETVVI greater than the median (P = .020). Neither PA/A nor eRV/eLV was associated with asthma severity or exacerbations.ConclusionsIn patients with asthma, smaller cardiac ventricular size may be associated with more severe disease and a higher rate of asthma exacerbations.Trial registryClinicalTrials.gov; No.: NCT01761630; URL: www.clinicaltrials.gov.

Combination of IL-6 (non-Type 2 asthma) and FeNO or blood eosinophil count (Type 2 asthma) identified asthma endotypes related to asthma severity, exacerbations, and responsiveness to corticosteroids and potential for response to anti-Type 2 and anti-IL-6 treatment.

BackgroundTools for quantification of asthma severity are limited.ObjectiveWe sought to develop a continuous measure of asthma severity, the Asthma Severity Scoring System (ASSESS), for adolescents and adults, incorporating domains of asthma control, lung function, medications, and exacerbations.MethodsBaseline and 36-month longitudinal data from participants in phase 3 of the Severe Asthma Research Program (NCT01606826) were used. Scale properties, responsiveness, and a minimally important difference were determined. External replication was performed in participants enrolled in the Severe Asthma Research Program phase 1/2. The utility of ASSESS for detecting treatment response was explored in participants undergoing corticosteroid responsiveness testing with intramuscular triamcinolone and participants receiving biologics.ResultsASSESS scores ranged from 0 to 20 (8.78 ± 3.9; greater scores reflect worse severity) and differed among 5 phenotypic groups. Measurement properties were acceptable. ASSESS was responsive to changes in quality of life with a minimally important difference of 2, with good specificity for outcomes of asthma improvement and worsening but poor sensitivity. Replication analyses yielded similar results, with a 2-point decrease (improvement) associated with improvements in quality of life. Participants with a 2-point or greater decrease (improvement) in ASSESS scores also had greater improvement in lung function and asthma control after triamcinolone, but these differences were limited to phenotypic clusters 3, 4, and 5. Participants treated with biologics also had a 2-point or greater decrease (improvement) in ASSESS scores overall.ConclusionsThe ASSESS tool is an objective measure that might be useful in epidemiologic and clinical research studies for quantification of treatment response in individual patients and phenotypic groups. However, validation studies are warranted.

BackgroundDespite advances in asthma care, disparities persist. Black patients are disproportionally affected by asthma and also have poorer outcomes compared with white patients.ObjectiveWe sought to determine associations between black and white patients and asthma-related health care use, accounting for complex relationships.MethodsThis study was completed as part of the National Heart, Lung, and Blood Institute's Severe Asthma Research Program, a prospective observational cohort. Between November 2012 and February 2015, it enrolled 579 participants 6 years and older with 1 year of observation time and complete data. Inverse probability of treatment weighting was used to balance racial groups with respect to community and family socioeconomic variables and environmental exposure variables. The primary outcome was emergency department (ED) use for asthma. Secondary outcomes included inhaled corticosteroid use, outpatient physician's office visits for asthma, and asthma-related hospitalization.ResultsBlack patients had greater odds of ED use over 1 year (odds ratio, 2.19; 95% CI, 1.43-3.35) but also differed in the majority (>50%) of baseline variables measured. After statistical balancing of the racial groups, the difference between black and white patients with respect to ED use no longer reached the level of significance. Instead, in secondary analyses black patients were less likely to see an outpatient physician for asthma management (adjusted odds ratio, 0.57; 95% CI, 0.38-0.85).ConclusionsThe disparity in ED use was eliminated after consideration of multiple variables. Social and environmental policies and interventions tailored to black populations with a high burden of asthma are critical to reduction (or elimination) of these disparities.

Rationale: Corticosteroids (CSs) are the most effective asthma therapy, but responses are heterogeneous and systemic CSs lead to long-term side effects. Therefore, an improved understanding of the contributing factors in CS responses could enhance precision management. Although several factors have been associated with CS responsiveness, no integrated/cluster approach has yet been undertaken to identify differential CS responses. Objectives: To identify asthma subphenotypes with differential responses to CS treatment using an unsupervised multiview learning approach. Methods: Multiple-kernel k-means clustering was applied to 100 clinical, physiological, inflammatory, and demographic variables from 346 adult participants with asthma in the Severe Asthma Research Program with paired (before and 2-3 weeks after triamcinolone administration) sputum data. Machine-learning techniques were used to select the top baseline variables that predicted cluster assignment for a new patient. Measurements and Main Results: Multiple-kernel clustering revealed four clusters of individuals with asthma and different CS responses. Clusters 1 and 2 consisted of young, modestly CS-responsive individuals with allergic asthma and relatively normal lung function, separated by contrasting sputum neutrophil and macrophage percentages after CS treatment. The subjects in cluster 3 had late-onset asthma and low lung function, high baseline eosinophilia, and the greatest CS responsiveness. Cluster 4 consisted primarily of young, obese females with severe airflow limitation, little eosinophilic inflammation, and the least CS responsiveness. The top 12 baseline variables were identified, and the clusters were validated using an independent Severe Asthma Research Program test set. Conclusions: Our machine learning-based approaches provide new insights into the mechanisms of CS responsiveness in asthma, with the potential to improve disease treatment.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1β secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1β in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values

BackgroundAirway type 2 inflammation is usually corticosteroid sensitive, but the role of type 2 inflammation as a mechanism of asthma in patients receiving high-dose inhaled corticosteroids (ICSs) is uncertain.ObjectiveWe sought to determine whether airway type 2 inflammation persists in patients treated with ICSs and to evaluate the clinical features of patients with steroid-resistant airway type 2 inflammation.MethodsWe used quantitative PCR to generate a composite metric of type 2 cytokine gene expression (type 2 gene mean [T2GM]) in induced sputum cells from healthy control subjects, patients with severe asthma receiving ICSs (n = 174), and patients with nonsevere asthma receiving ICSs (n = 85). We explored relationships between asthma outcomes and T2GM values and the utility of noninvasive biomarkers of airway T2GM.ResultsSputum cell T2GM values in asthmatic patients were significantly increased and remained high after treatment with intramuscular triamcinolone. We used the median T2GM value as a cutoff to classify steroid-treated type 2-low and steroid-resistant type 2-high (srT2-high) subgroups. Compared with patients with steroid-treated type 2-low asthma, those with srT2-high asthma were older and had more severe asthma. Blood eosinophil cell counts predicted srT2-high asthma when body mass index was less than 40 kg/m2 but not when it was 40 kg/m2 or greater, whereas blood IgE levels strongly predicted srT2-high asthma when age was less than 34 years but not when it was 34 years or greater.ConclusionDespite ICS therapy, many asthmatic patients have persistent airway type 2 inflammation (srT2-high asthma), and these patients are older and have more severe disease. Body weight and age modify the performance of blood-based biomarkers of airway type 2 inflammation.

Severe asthma is a debilitating and treatment refractory disease. As many as half of these patients have complex neutrophil-predominant lung inflammation that is distinct from milder asthma with type 2 eosinophilic inflammation. New insights into severe asthma pathogenesis are needed. Concomitant exposure of mice to an aeroallergen and endotoxin during sensitization resulted in complex neutrophilic immune responses to allergen alone during later airway challenge. Unlike allergen alone, sensitization with allergen and endotoxin led to NETosis. In addition to neutrophil extracellular traps (NETs), enucleated neutrophil cytoplasts were evident in the lungs. Surprisingly, allergen-driven airway neutrophilia was decreased in peptidyl arginine deiminase 4-deficient mice with defective NETosis but not by deoxyribonuclease treatment, implicating the cytoplasts for the non-type 2 immune responses to allergen. Neutrophil cytoplasts were also present in mediastinal lymph nodes, and the cytoplasts activated lung dendritic cells in vitro to trigger antigen-specific interleukin-17 (IL-17) production from naïve CD4+ T cells. Bronchoalveolar lavage fluid from patients with severe asthma and high neutrophil counts had detectable NETs and cytoplasts that were positively correlated with IL-17 levels. Together, these translational findings have identified neutrophil cytoplast formation in asthmatic lung inflammation and linked the cytoplasts to T helper 17-mediated neutrophilic inflammation in severe asthma.

RationaleLoss of the peripheral pulmonary vasculature, termed vascular pruning, is associated with disease severity in patients with chronic obstructive pulmonary disease.ObjectivesTo determine if pulmonary vascular pruning is associated with asthma severity and exacerbations.MethodsWe measured the total pulmonary blood vessel volume (TBV) and the blood vessel volume of vessels less than 5 mm2 in cross-sectional area (BV5) and of vessels less than 10 mm2 (BV10) in cross-sectional area on noncontrast computed tomographic scans of participants from the Severe Asthma Research Program. Lower values of the BV5 to TBV ratio (BV5/TBV) and the BV10 to TBV ratio (BV10/TBV) represented vascular pruning (loss of the peripheral pulmonary vasculature).Measurements and main resultsCompared with healthy control subjects, patients with severe asthma had more pulmonary vascular pruning. Among those with asthma, those with poor asthma control had more pruning than those with well-controlled disease. Pruning of the pulmonary vasculature was also associated with lower percent predicted FEV1 and FVC, greater peripheral and sputum eosinophilia, and higher BAL serum amyloid A/lipoxin A4 ratio but not with low-attenuation area or with sputum neutrophilia. Compared with individuals with less pruning, individuals with the most vascular pruning had 150% greater odds of reporting an asthma exacerbation (odds ratio, 2.50; confidence interval, 1.05-5.98; P = 0.039 for BV10/TBV) and reported 45% more asthma exacerbations during follow-up (incidence rate ratio, 1.45; confidence interval, 1.02-2.06; P = 0.036 for BV10/TBV).ConclusionsPruning of the peripheral pulmonary vasculature is associated with asthma severity, control, and exacerbations, and with lung function and eosinophilia.

BackgroundThe link between mucus plugs and airflow obstruction has not been established in chronic severe asthma, and the role of eosinophils and their products in mucus plug formation is unknown.MethodsIn clinical studies, we developed and applied a bronchopulmonary segment-based scoring system to quantify mucus plugs on multidetector computed tomography (MDCT) lung scans from 146 subjects with asthma and 22 controls, and analyzed relationships among mucus plug scores, forced expiratory volume in 1 second (FEV1), and airway eosinophils. Additionally, we used airway mucus gel models to explore whether oxidants generated by eosinophil peroxidase (EPO) oxidize cysteine thiol groups to promote mucus plug formation.ResultsMucus plugs occurred in at least 1 of 20 lung segments in 58% of subjects with asthma and in only 4.5% of controls, and the plugs in subjects with asthma persisted in the same segment for years. A high mucus score (plugs in ≥ 4 segments) occurred in 67% of subjects with asthma with FEV1 of less than 60% of predicted volume, 19% with FEV1 of 60%-80%, and 6% with FEV1 greater than 80% (P < 0.001) and was associated with marked increases in sputum eosinophils and EPO. EPO catalyzed oxidation of thiocyanate and bromide by H2O2 to generate oxidants that crosslink cysteine thiol groups and stiffen thiolated hydrogels.ConclusionMucus plugs are a plausible mechanism of chronic airflow obstruction in severe asthma, and EPO-generated oxidants may mediate mucus plug formation. We propose an approach for quantifying airway mucus plugging using MDCT lung scans and suggest that treating mucus plugs may improve airflow in chronic severe asthma.Trial registrationClinicaltrials.gov NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01759186, NCT01716494, and NCT01760915.FundingNIH grants P01 HL107201, R01 HL080414, U10 HL109146, U10 HL109164, U10 HL109172, U10 HL109086, U10 HL109250, U10 HL109168, U10 HL109257, U10 HL109152, and P01 HL107202 and National Center for Advancing Translational Sciences grants UL1TR0000427, UL1TR000448, and KL2TR000428.

In health, inflammation resolution is an active process governed by specialized proresolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both proresolving and proinflammatory ligands for opposing signaling events, suggesting pivotal roles for ALX in the fate of inflammatory responses. Here, we determined if ALX expression and ligands were linked to severe asthma (SA). ALX expression and levels of proresolving ligands (lipoxin A4 [LXA4], 15-epi-LXA4, and annexin A1 [ANXA1]), and a proinflammatory ligand (serum amyloid A [SAA]) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA [n = 69], non-SA [NSA, n = 51] or healthy donors [HDs, n = 47]). Bronchoalveolar lavage (BAL) fluid LXA4 and 15-epi-LXA4 were decreased and SAA was increased in SA relative to NSA. BAL macrophage ALX expression was increased in SA. Subjects with LXA4loSAAhi levels had increased BAL neutrophils, more asthma symptoms, lower lung function, increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease, and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA4loSAAhi BAL fluid induced IL-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by coincubation with 15-epi-LXA4. Together, these findings have established an association between select ALX receptor ligands and asthma severity that define a potentially new biochemical endotype for asthma and support a pivotal functional role for ALX signaling in the fate of lung inflammation. Severe Asthma Research Program-3 (SARP-3; ClinicalTrials.gov NCT01606826)FUNDING Sources. National Heart, Lung and Blood Institute, the NIH, and the German Society of Pediatric Pneumology.

RationalePhenotypic distinctions between severe asthma (SA) and nonsevere asthma (NONSA) may be confounded by differential adherence or incorrect use of corticosteroids.ObjectivesTo determine if there are persistent phenotypic distinctions between SA (as defined by 2014 American Thoracic Society/European Respiratory Society guidelines) and NONSA after intramuscular triamcinolone acetonide (TA), and to identify predictors of a corticosteroid response in these populations.MethodsA total of 526 adults age 18 years and older (315 SA) and 188 children age 6 to less than 18 years (107 SA) in the NHLBI Severe Asthma Research Program III were characterized before and 3 weeks after TA. The primary outcome for corticosteroid response was defined as greater than or equal to 10-point improvement in percent predicted FEV1.Measurements and main resultsAdult asthma groups exhibited a small but significant mean FEV1% predicted improvement after TA (SA group mean difference, 3.4%; 95% confidence interval, 2.2-4.7%; P = 0.001), whereas children did not. Adult SA continued to manifest lower FEV1 and worse asthma control as compared with NONSA after TA. In children, after TA only prebronchodilator FEV1 distinguished SA from NONSA. A total of 21% of adults with SA and 20% of children with SA achieved greater than or equal to 10% improvement after TA. Baseline bronchodilator response and fractional exhaled nitric oxide had good sensitivity and specificity for predicting response in all groups except children with NONSA.ConclusionsOne in five patients with SA exhibit greater than or equal to 10% improvement in FEV1 with parenteral corticosteroid. Those likely to respond had greater bronchodilator responsiveness and fractional exhaled nitric oxide levels. In adults, differences in airflow obstruction and symptoms between SA and NONSA persist after parenteral corticosteroids, suggesting a component of corticosteroid nonresponsive pathobiology in adults with SA that may differ in children. Clinical trial registered with www.clinicaltrials.gov (NCT 01606826).

Severe asthma is typically characterized by chronic airway inflammation that is refractory to corticosteroids and associated with excess morbidity. Patients were recruited into the National Heart, Lung, and Blood Institute-sponsored Severe Asthma Research Program and comprehensively phenotyped by bronchoscopy. Bronchoalveolar lavage (BAL) cells were analyzed by flow cytometry. Compared with healthy individuals (n = 21), patients with asthma (n = 53) had fewer BAL natural killer (NK) cells. Patients with severe asthma (n = 29) had a marked increase in the ratios of CD4+ T cells to NK cells and neutrophils to NK cells. BAL NK cells in severe asthma were skewed toward the cytotoxic CD56dim subset, with significantly increased BAL fluid levels of the cytotoxic mediator granzyme A. The numbers of BAL CD56dim NK cells and CCR6-CCR4- T helper 1-enriched CD4+ T cells correlated inversely with lung function [forced expiratory volume in 1 s (FEV1) % predicted] in asthma. Relative to cells from healthy controls, peripheral blood NK cells from asthmatic patients had impaired killing of K562 myeloid target cells despite releasing more cytotoxic mediators. Ex vivo exposure to dexamethasone markedly decreased blood NK cell lysis of target cells and cytotoxic mediator release. NK cells expressed airway lipoxin A4/formyl peptide receptor 2 receptors, and in contrast to dexamethasone, lipoxin A4-exposed NK cells had preserved functional responses. Together, our findings indicate that the immunology of the severe asthma airway is characterized by decreased NK cell cytotoxicity with increased numbers of target leukocytes, which is exacerbated by corticosteroids that further disable NK cell function. These failed resolution mechanisms likely contribute to persistent airway inflammation in severe asthma.

RationaleReducing asthma exacerbation frequency is an important criterion for approval of asthma therapies, but the clinical features of exacerbation-prone asthma (EPA) remain incompletely defined.ObjectivesTo describe the clinical, physiologic, inflammatory, and comorbidity factors associated with EPA.MethodsBaseline 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 resultsOf 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.ConclusionsEPA may be a distinct susceptibility phenotype with implications for the targeting of exacerbation prevention strategies. Clinical trial registered with www.clinicaltrials.gov (NCT 01760915).

Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8-driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.

BackgroundSevere 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.MethodsIn 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.FindingsBetween 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

Asthma is a common chronic disease without cure. Our understanding of asthma onset, pathobiology, classification, and management has evolved substantially over the past decade; however, significant asthma-related morbidity and excess healthcare use and costs persist. To address this important clinical condition, the NHLBI convened a group of extramural investigators for an Asthma Research Strategic Planning workshop on September 18-19, 2014, to accelerate discoveries and their translation to patients. The workshop focused on (1) in utero and early-life origins of asthma, (2) the use of phenotypes and endotypes to classify disease, (3) defining disease modification, (4) disease management, and (5) implementation research. This report summarizes the workshop and produces recommendations to guide future research in asthma.

Previous studies demonstrated that bone marrow-derived mesenchymal stem (stromal) cells (MSCs) reduce the severity of acute lung injury in animal models and in an ex vivo perfused human lung model. However, the mechanisms by which MSCs reduce lung injury are not well understood. In the present study, we tested the hypothesis that human MSCs promote the resolution of acute lung injury in part through the effects of a specialized proresolving mediator lipoxin A4 (LXA4). Human alveolar epithelial type II cells and MSCs expressed biosynthetic enzymes and receptors for LXA4. Coculture of human MSCs with alveolar epithelial type II cells in the presence of cytomix significantly increased the production of LXA4 by 117%. The adoptive transfer of MSCs after the onset of LPS-induced acute lung injury (ALI) in mice led to improved survival (48 h), and blocking the LXA4 receptor with WRW4, a LXA4 receptor antagonist, significantly reversed the protective effect of MSCs on both survival and the accumulation of pulmonary edema. LXA4 alone improved survival in mice, and it also significantly decreased the production of TNF-α and MIP-2 in bronchoalveolar lavage fluid. In summary, these experiments demonstrated two novel findings: human MSCs promote the resolution of lung injury in mice in part through the proresolving lipid mediator LXA4, and LXA4 itself should be considered as a therapeutic for acute respiratory distress syndrome.

The aging of the population in the United States and throughout the developed world has increased morbidity and mortality attributable to lung disease, while the morbidity and mortality from other prevalent diseases has declined or remained stable. Recognizing the importance of aging in the development of lung disease, the American Thoracic Society (ATS) highlighted this topic as a core theme for the 2014 annual meeting. The relationship between aging and lung disease was discussed in several oral symposiums and poster sessions at the annual ATS meeting. In this article, we used the input gathered at the conference to develop a broad framework and perspective to stimulate basic, clinical, and translational research to understand how the aging process contributes to the onset and/or progression of lung diseases. A consistent theme that emerged from the conference was the need to apply novel, systems-based approaches to integrate a growing body of genomic, epigenomic, transcriptomic, and proteomic data and elucidate the relationship between biologic hallmarks of aging, altered lung function, and increased susceptibility to lung diseases in the older population. The challenge remains to causally link the molecular and cellular changes of aging with age-related changes in lung physiology and disease susceptibility. The purpose of this review is to stimulate further research to identify new strategies to prevent or treat age-related lung disease.

RationaleSevere asthma is characterized by airway inflammatory responses associated with aberrant metabolism of arachidonic acid. Lipoxins (LX) are arachidonate-derived pro-resolving mediators that are decreased in severe asthma, yet mechanisms for defective LX biosynthesis and a means to increase LXs in severe asthma remain to be established.ObjectivesTo determine if oxidative stress and soluble epoxide hydrolase (sEH) activity are linked to decreased LX biosynthesis in severe asthma.MethodsAliquots of blood, sputum, and bronchoalveolar lavage fluid were obtained from asthma subjects for mediator determination. Select samples were exposed to t-butyl-hydroperoxide or sEH inhibitor (sEHI) before activation. Peripheral blood leukocyte-platelet aggregates were monitored by flow cytometry, and bronchial contraction was determined with cytokine-treated human lung sections.Measurements and main results8-Isoprostane levels in sputum supernatants were inversely related to LXA4 in severe asthma (r = -0.55; P = 0.03) and t-butyl-hydroperoxide decreased LXA4 and 15-epi-LXA4 biosynthesis by peripheral blood leukocytes. LXA4 and 15-epi-LXA4 levels were inversely related to sEH activity in sputum supernatants and sEHIs significantly increased 14,15-epoxy-eicosatrienoic acid and 15-epi-LXA4 generation by severe asthma whole blood and bronchoalveolar lavage fluid cells. The abundance of peripheral blood leukocyte-platelet aggregates was related to asthma severity. In a concentration-dependent manner, LXs significantly inhibited platelet-activating factor-induced increases in leukocyte-platelet aggregates (70.8% inhibition [LXA4 100 nM], 78.3% inhibition [15-epi-LXA4 100 nM]) and 15-epi-LXA4 markedly inhibited tumor necrosis factor-α-induced increases in bronchial contraction.ConclusionsLX levels were decreased by oxidative stress and sEH activity. Inhibitors of sEH increased LXs that mediated antiphlogistic actions, suggesting a new therapeutic approach for severe asthma. Clinical trial registered with www.clinicaltrials.gov (NCT 00595114).