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BackgroundImproved understanding of the pathways associated with airway pathophysiologic features in COPD will identify new predictive biomarkers and novel therapeutic targets.Research questionWhich physiologic pathways are altered in the airways of patients with COPD and will predict exacerbations?Study design and methodsWe applied a mass spectrometric panel of metabolomic biomarkers related to mucus hydration and inflammation to sputa from the multicenter Subpopulations and Intermediate Outcome Measures in COPD Study. Biomarkers elevated in sputa from patients with COPD were evaluated for relationships to measures of COPD disease severity and their ability to predict future exacerbations.ResultsSputum supernatants from 980 patients were analyzed: 77 healthy nonsmokers, 341 smokers with preserved spirometry, and 562 patients with COPD (178 with Global Initiative on Chronic Obstructive Lung Disease [GOLD] stage 1 disease, 303 with GOLD stage 2 disease, and 81 with GOLD stage 3 disease) were analyzed. Biomarkers from multiple pathways were elevated in COPD and correlated with sputum neutrophil counts. Among the most significant analytes (false discovery rate, 0.1) were sialic acid, hypoxanthine, xanthine, methylthioadenosine, adenine, and glutathione. Sialic acid and hypoxanthine were associated strongly with measures of disease severity, and elevation of these biomarkers was associated with shorter time to exacerbation and improved prediction models of future exacerbations.InterpretationBiomarker evaluation implicated pathways involved in mucus hydration, adenosine metabolism, methionine salvage, and oxidative stress in COPD airway pathophysiologic characteristics. Therapies that target these pathways may be of benefit in COPD, and a simple model adding sputum-soluble phase biomarkers improves prediction of pulmonary exacerbations.Trial registryClinicalTrials.gov; No.: NCT01969344; URL: www.Clinicaltrialsgov.

BackgroundWhether microbiome characteristics of induced sputum or oral samples demonstrate unique relationships to features of atopy or mild asthma in adults is unknown.ObjectiveWe sought to determine sputum and oral microbiota relationships to clinical or immunologic features in mild atopic asthma and the impact on the microbiota of inhaled corticosteroid (ICS) treatment administered to ICS-naive subjects with asthma.MethodsBacterial microbiota profiles were analyzed in induced sputum and oral wash samples from 32 subjects with mild atopic asthma before and after inhaled fluticasone treatment, 18 atopic subjects without asthma, and 16 nonatopic healthy subjects in a multicenter study (NCT01537133). Associations with clinical and immunologic features were examined, including markers of atopy, type 2 inflammation, immune cell populations, and cytokines.ResultsSputum bacterial burden inversely associated with bronchial expression of type 2 (T2)-related genes. Differences in specific sputum microbiota also associated with T2-low asthma phenotype, a subgroup of whom displayed elevations in lung inflammatory mediators and reduced sputum bacterial diversity. Differences in specific oral microbiota were more reflective of atopic status. After ICS treatment of patients with asthma, the compositional structure of sputum microbiota showed greater deviation from baseline in ICS nonresponders than in ICS responders.ConclusionsNovel associations of sputum and oral microbiota to immunologic features were observed in this cohort of subjects with or without ICS-naive mild asthma. These findings confirm and extend our previous report of reduced bronchial bacterial burden and compositional complexity in subjects with T2-high asthma, with additional identification of a T2-low subgroup with a distinct microbiota-immunologic relationship.

BackgroundPerturbations to the composition and function of bronchial bacterial communities appear to contribute to the pathophysiology of asthma. Unraveling the nature and mechanisms of these complex associations will require large longitudinal studies, for which bronchoscopy is poorly suited. Studies of samples obtained by sputum induction and nasopharyngeal brushing or lavage have also reported asthma-associated microbiota characteristics. It remains unknown, however, whether the microbiota detected in these less-invasive sample types reflect the composition of bronchial microbiota in asthma.ResultsBacterial microbiota in paired protected bronchial brushings (BB; n = 45), induced sputum (IS; n = 45), oral wash (OW; n = 45), and nasal brushings (NB; n = 27) from adults with mild atopic asthma (AA), atopy without asthma (ANA), and healthy controls (HC) were profiled using 16S rRNA gene sequencing. Though microbiota composition varied with sample type (p

BackgroundCompositional differences in the bronchial bacterial microbiota have been associated with asthma, but it remains unclear whether the findings are attributable to asthma, to aeroallergen sensitization, or to inhaled corticosteroid treatment.ObjectivesWe sought to compare the bronchial bacterial microbiota in adults with steroid-naive atopic asthma, subjects with atopy but no asthma, and nonatopic healthy control subjects and to determine relationships of the bronchial microbiota to phenotypic features of asthma.MethodsBacterial communities in protected bronchial brushings from 42 atopic asthmatic subjects, 21 subjects with atopy but no asthma, and 21 healthy control subjects were profiled by using 16S rRNA gene sequencing. Bacterial composition and community-level functions inferred from sequence profiles were analyzed for between-group differences. Associations with clinical and inflammatory variables were examined, including markers of type 2-related inflammation and change in airway hyperresponsiveness after 6 weeks of fluticasone treatment.ResultsThe bronchial microbiome differed significantly among the 3 groups. Asthmatic subjects were uniquely enriched in members of the Haemophilus, Neisseria, Fusobacterium, and Porphyromonas species and the Sphingomonodaceae family and depleted in members of the Mogibacteriaceae family and Lactobacillales order. Asthma-associated differences in predicted bacterial functions included involvement of amino acid and short-chain fatty acid metabolism pathways. Subjects with type 2-high asthma harbored significantly lower bronchial bacterial burden. Distinct changes in specific microbiota members were seen after fluticasone treatment. Steroid responsiveness was linked to differences in baseline compositional and functional features of the bacterial microbiome.ConclusionEven in subjects with mild steroid-naive asthma, differences in the bronchial microbiome are associated with immunologic and clinical features of the disease. The specific differences identified suggest possible microbiome targets for future approaches to asthma treatment or prevention.

In the present studies, the assessment of how viral exacerbation of asthmatic responses with and without pulmonary steroid treatment alters the microbiome in conjunction with immune responses presents striking data. The overall findings identify that although steroid treatment of allergic animals diminished the severity of the respiratory syncytial virus (RSV)-induced exacerbation of airway function and mucus hypersecretion, there were local increases in IL-17 expression. Analysis of the lung and gut microbiome suggested that there are differences in RSV exacerbation that are further altered by fluticasone (FLUT) treatment. Using metagenomic inference software, PICRUSt2, we were able to predict that the metabolite profile produced by the changed gut microbiome was significantly different with multiple metabolic pathways and associated with specific treatments with or without FLUT. Importantly, measuring plasma metabolites in an unbiased manner, our data indicate that there are significant changes associated with chronic allergen exposure, RSV exacerbation, and FLUT treatment that are reflective of responses to the disease and treatment. In addition, the changes in metabolites appeared to have contributions from both host and microbial pathways. To understand if airway steroids on their own altered lung and gut microbiome along with host responses to RSV infection, naïve animals were treated with lung FLUT before RSV infection. The naïve animals treated with FLUT before RSV infection demonstrated enhanced disease that corresponded to an altered microbiome and the related PICRUSt2 metagenomic inference analysis. Altogether, these findings set the foundation for identifying important correlations of severe viral exacerbated allergic disease with microbiome changes and the relationship of host metabolome with a potential for early life pulmonary steroid influence on subsequent viral-induced disease. NEW & NOTEWORTHY: These studies outline a novel finding that airway treatment with fluticasone, a commonly used inhaled steroid, has significant effects on not only the local lung environment but also on the mucosal microbiome, which may have significant disease implications. The findings further provide data to support that pulmonary viral exacerbations of asthma with or without steroid treatment alter the lung and gut microbiome, which have an impact on the circulating metabolome that likely alters the trajectory of disease progression. [ABSTRACT FROM AUTHOR]

BackgroundAsthma is heterogeneous, and airway dysbiosis is associated with clinical features in patients with mild-to-moderate asthma. Whether similar relationships exist among patients with severe asthma is unknown.ObjectiveWe sought to evaluate relationships between the bronchial microbiome and features of severe asthma.MethodsBronchial brushings from 40 participants in the Bronchoscopic Exploratory Research Study of Biomarkers in Corticosteroid-refractory Asthma (BOBCAT) study were evaluated by using 16S ribosomal RNA-based methods. Relationships to clinical and inflammatory features were analyzed among microbiome-profiled subjects. Secondarily, bacterial compositional profiles were compared between patients with severe asthma and previously studied healthy control subjects (n = 7) and patients with mild-to-moderate asthma (n = 41).ResultsIn patients with severe asthma, bronchial bacterial composition was associated with several disease-related features, including body mass index (P < .05, Bray-Curtis distance-based permutational multivariate analysis of variance; PERMANOVA), changes in Asthma Control Questionnaire (ACQ) scores (P < .01), sputum total leukocyte values (P = .06), and bronchial biopsy eosinophil values (per square millimeter, P = .07). Bacterial communities associated with worsening ACQ scores and sputum total leukocyte values (predominantly Proteobacteria) differed markedly from those associated with body mass index (Bacteroidetes/Firmicutes). In contrast, improving/stable ACQ scores and bronchial epithelial gene expression of FK506 binding protein (FKBP5), an indicator of steroid responsiveness, correlated with Actinobacteria. Mostly negative correlations were observed between biopsy eosinophil values and Proteobacteria. No taxa were associated with a TH2-related epithelial gene expression signature, but expression of TH17-related genes was associated with Proteobacteria. Patients with severe asthma compared with healthy control subjects or patients with mild-to-moderate asthma were significantly enriched in Actinobacteria, although the largest differences observed involved a Klebsiella genus member (7.8-fold increase in patients with severe asthma, adjusted P < .001).ConclusionsSpecific microbiota are associated with and may modulate inflammatory processes in patients with severe asthma and related phenotypes. Airway dysbiosis in patients with severe asthma appears to differ from that observed in those with milder asthma in the setting of inhaled corticosteroid use.

That the subglottic airways are not sterile, as was once believed, but are populated by a distinct "bronchial microbiome," is now accepted. Also accepted is the concept that asthma is associated with differences in the composition of this microbiome. What is not clear is whether the differences in microbial community composition themselves mediate pathologic changes in the airways or whether they reflect differences in systemic immune function driven by differences in the development of the gastrointestinal microbiome in early life, when the immune system is most malleable. Recognition of the probable existence of a "common mucosal immune system" allowed synthesis of these apparently opposing ideas into a single conceptual model. Gastrointestinal microbiome-driven differences in systemic immune function predispose to sensitization to allergens deposited on mucosal surfaces, whereas possibly similar, but not identical, differences in immune function predispose to less effective responses to microbial infection of the airways, resulting in persistence of the inflammation underlying the structural and functional abnormalities of asthma. In this model, allergic sensitization and asthma are thus seen as commonly overlapping but not necessarily coincident consequences of abnormalities in microbial colonization, development of immune function, and encounter with agents infecting the respiratory tract.

Specific bacterial species are implicated in the pathogenesis of exacerbations of chronic obstructive pulmonary disease (COPD). However, recent studies of clinically stable COPD patients have demonstrated a greater diversity of airway microbiota, whose role in acute exacerbations is unclear. In this study, temporal changes in the airway microbiome before, at the onset of, and after an acute exacerbation were examined in 60 sputum samples collected from subjects enrolled in a longitudinal study of bacterial infection in COPD. Microbiome composition and predicted functions were examined using 16S rRNA-based culture-independent profiling methods. Shifts in the abundance (≥ 2-fold, P < 0.05) of many taxa at exacerbation and after treatment were observed. Microbiota members that were increased at exacerbation were primarily of the Proteobacteria phylum, including nontypical COPD pathogens. Changes in the bacterial composition after treatment for an exacerbation differed significantly among the therapy regimens clinically prescribed (antibiotics only, oral corticosteroids only, or both). Treatment with antibiotics alone primarily decreased the abundance of Proteobacteria, with the prolonged suppression of some microbiota members being observed. In contrast, treatment with corticosteroids alone led to enrichment for Proteobacteria and members of other phyla. Predicted metagenomes of particular microbiota members involved in these compositional shifts indicated exacerbation-associated loss of functions involved in the synthesis of antimicrobial and anti-inflammatory products, alongside enrichment in functions related to pathogen-elicited inflammation. These trends reversed upon clinical recovery. Further larger studies will be necessary to determine whether specific compositional or functional changes detected in the airway microbiome could be useful indicators of exacerbation development or outcome.

That the subglottic airways are not sterile, as was once believed, but are populated by a distinct "bronchial microbiome," is now accepted. Also accepted is the concept that asthma is associated with differences in the composition of this microbiome. What is not clear is whether the differences in microbial community composition themselves mediate pathologic changes in the airways or whether they reflect differences in systemic immune function driven by differences in the development of the gastrointestinal microbiome in early life, when the immune system is most malleable. Recognition of the probable existence of a "common mucosal immune system" allowed synthesis of these apparently opposing ideas into a single conceptual model. Gastrointestinal microbiome-driven differences in systemic immune function predispose to sensitization to allergens deposited on mucosal surfaces, whereas possibly similar, but not identical, differences in immune function predispose to less effective responses to microbial infection of the airways, resulting in persistence of the inflammation underlying the structural and functional abnormalities of asthma. In this model, allergic sensitization and asthma are thus seen as commonly overlapping but not necessarily coincident consequences of abnormalities in microbial colonization, development of immune function, and encounter with agents infecting the respiratory tract.

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective "early" and "late" colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.

Colonization of the infant gut by microorganisms over the first year of life is crucial for development of a balanced immune response. Early alterations in the gastrointestinal microbiota of neonates has been linked with subsequent development of asthma and atopy in older children. Here we describe high-resolution culture-independent analysis of stool samples from 6-month old infants fed daily supplements of Lactobacillus casei subsp. Rhamnosus (LGG) or placebo in a double-blind, randomized Trial of Infant Probiotic Supplementation (TIPS). Bacterial community composition was examined using a high-density microarray, the 16S rRNA PhyloChip, and the microbial assemblages of infants with either high or low LGG abundance were compared. Communities with high abundance of LGG exhibited promotion of phylogenetically clustered taxa including a number of other known probiotic species, and were significantly more even in their distribution of community members. Ecologically, these aspects are characteristic of communities that are more resistant to perturbation and outgrowth of pathogens. PhyloChip analysis also permitted identification of taxa negatively correlated with LGG abundance that have previously been associated with atopy, as well as those positively correlated that may prove useful alternative targets for investigation as alternative probiotic species. From these findings we hypothesize that a key mechanism for the protective effect of LGG supplementation on subsequent development of allergic disease is through promotion of a stable, even, and functionally redundant infant gastrointestinal community.

Rationale: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPDs) are associated with a significant disease burden. Blood immune phenotyping may improve our understanding of a COPD endotype at increased risk of exacerbations. Objective: To determine the relationship between the transcriptome of circulating leukocytes and COPD exacerbations. Methods: Blood RNA sequencing data (n = 3,618) from the COPDGene (Genetic Epidemiology of COPD) study were analyzed. Blood microarray data (n = 646) from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study were used for validation. We tested the association between blood gene expression and AE-COPDs. We imputed the abundance of leukocyte subtypes and tested their association with prospective AE-COPDs. Flow cytometry was performed on blood in SPIROMICS (Subpopulations and Intermediate Outcomes in COPD Study) (n = 127), and activation markers for T cells were tested for association with prospective AE-COPDs. Measurements and Main Results: Exacerbations were reported 4,030 and 2,368 times during follow-up in COPDGene (5.3 ± 1.7 yr) and ECLIPSE (3 yr), respectively. We identified 890, 675, and 3,217 genes associated with a history of AE-COPDs, persistent exacerbations (at least one exacerbation per year), and prospective exacerbation rate, respectively. In COPDGene, the number of prospective exacerbations in patients with COPD (Global Initiative for Chronic Obstructive Lung Disease stage ⩾2) was negatively associated with circulating CD8+ T cells, CD4+ T cells, and resting natural killer cells. The negative association with naive CD4+ T cells was replicated in ECLIPSE. In the flow-cytometry study, an increase in CTLA4 on CD4+ T cells was positively associated with AE-COPDs. Conclusions: Individuals with COPD with lower circulating lymphocyte counts, particularly decreased CD4+ T cells, are more susceptible to AE-COPDs, including persistent exacerbations. [ABSTRACT FROM AUTHOR]