Human genetic associations of the airway microbiome in chronic obstructive pulmonary disease.

Little is known about the relationships between human genetics and the airway microbiome. Deeply sequenced airway metagenomics, by simultaneously characterizing the microbiome and host genetics, provide a unique opportunity to assess the microbiome-host genetic associations. Here we performed a co-profiling of microbiome and host genetics with the identification of over 5 million single nucleotide polymorphisms (SNPs) through deep metagenomic sequencing in sputum of 99 chronic obstructive pulmonary disease (COPD) and 36 healthy individuals. Host genetic variation was the most significant factor associated with the microbiome except for geography and disease status, with its top 5 principal components accounting for 12.11% of the microbiome variability. Within COPD individuals, 113 SNPs mapped to candidate genes reported as genetically associated with COPD exhibited associations with 29 microbial species and 48 functional modules (P < 1 × 10-5), where Streptococcus salivarius exhibits the strongest association to SNP rs6917641 in TBC1D32 (P = 9.54 × 10-8). Integration of concurrent host transcriptomic data identified correlations between the expression of host genes and their genetically-linked microbiome features, including NUDT1, MAD1L1 and Veillonella parvula, TTLL9 and Stenotrophomonas maltophilia, and LTA4H and Haemophilus influenzae. Mendelian randomization analyses revealed a potential causal link between PARK7 expression and microbial type III secretion system, and a genetically-mediated association between COPD and increased relative abundance of airway Streptococcus intermedius. These results suggest a previously underappreciated role of host genetics in shaping the airway microbiome and provide fresh hypotheses for genetic-based host-microbiome interactions in COPD.

Low human beta-defensin-2 levels in the sputum of COPD patients are associated with the risk of exacerbations.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is a complicated chronic inflammatory disease. It is important to investigate the characteristics of acute exacerbation of COPD to develop new therapeutic strategies. OBJECTIVE: This study aimed to determine the relationship between the human beta-defensin-2 (hBD-2) levels and aggravation of COPD. METHODS: We detected the sputum hBD-2 level of 254 patients from Guangzhou, China, for 2 years. The study participants were categorized into the COPD group (n = 203, GOLD 0-4) and the control group (n = 51, 40-79 years old). At baseline, 12th month, and 24th month, we detected the sputum hBD-2 level and levels of cytokines, such as CXCL10, CXCL11, and IFN. RESULTS: At baseline, there were no significant differences in the sputum and serum hBD-2 levels between the patients and the controls. However, the sputum hBD-2 levels of patients who had at least one symptom aggravation over the next 2 years were significantly lower than those of patients without any exacerbations (1130.9 ± 858.4 pg/mL vs. 2103.7 ± 1294.2 pg/mL, respectively; p = 0.001). Nevertheless, there were no statistically significant differences in the sputum hBD-2 levels between patients (no aggravation history) and controls (2084.9 ± 1317.6 pg/mL vs. 2152.5 ± 1251.6 pg/mL, respectively; p = 0.626). We used a logistic regression model to assess the relationship between aggravation and sputum hBD-2 levels. Interestingly, we found that low hBD-2 level (< 1000 pg/mL) was significantly associated with exacerbations. Specifically, patients with low hBD-2 levels were more likely to experience exacerbations in the next 12 months (0.333 vs. 0.117; p = 0.001). Moreover, we compared the hBD-2 levels between controls and patients with GOLD 3-4 and found that participants with bacteria (+) and/or viruses (+) had an association between hBD-2 level and disease severity (p = 0.02). CONCLUSION: Patients at risk of exacerbations are more likely to have lower sputum hBD-2 levels. These results have important implications for future therapies for COPD.

Correlations of Computed Tomography Measurement of Distal Pulmonary Vascular Pruning with Airflow Limitation and Emphysema in COPD Patients.

Background: Pulmonary vascular alteration is an important feature of chronic obstructive pulmonary disease (COPD), which is characterized by distal pulmonary vascular pruning in angiography. We aimed to further investigate the clinical relevance of pulmonary vasculature in COPD patients using non-contrast computed tomography (CT). Methods: Seventy-one control subjects and 216 COPD patients completed the questionnaires, spirometry, and computed tomography (CT) scans within 1 month and were included in the study. Small pulmonary vessels represented by percentage of cross-sectional area of pulmonary vessels smaller than 5 mm2 or 5-10 mm2 to the total lung fields (%CSA<5 or %CSA5-10, respectively) were measured using ImageJ software. Spearman correlation was used to investigate the relationship between %CSA<5 and airflow limitation. A receiver operating characteristic (ROC) curve was built to evaluate the value of %CSA<5 in discriminating COPD patients from healthy control subjects. Segmented regression was used to analyze the relationship between %CSA<5 and %LAA-950 (percentage of low-attenuation areas less than -950 HU). Results: We found a significant correlation between %CSA<5 and forced expiratory volume in one second (FEV1) percentage of predicted value (%pred) (r = 0.564, P < 0.001). The area under the ROC curve for the value of %CSA<5 in distinguishing COPD was 0.816, with a cut-off value of 0.537 (Youden index J, 0.501; sensitivity, 78.24%; specificity, 71.83%). Since the relationship between %CSA<5 and %LAA-950 was not constant, performance of segmented regression was better than ordinary linear regression (adjusted R2, 0.474 vs 0.332, P < 0.001 and P < 0.001, respectively). As %CSA<5 decreased, %LAA-950 slightly increased until an inflection point (%CSA<5 = 0.524) was reached, after which the %LAA-950 increased apparently with a decrease in %CSA<5. Conclusion: %CSA<5 was significantly correlated with both airflow limitation and emphysema, and we identified an inflection point for the relationship between %CSA<5 and %LAA-950.

Multi-omics analyses of airway host-microbe interactions in chronic obstructive pulmonary disease identify potential therapeutic interventions.

The mechanistic role of the airway microbiome in chronic obstructive pulmonary disease (COPD) remains largely unexplored. We present a landscape of airway microbe-host interactions in COPD through an in-depth profiling of the sputum metagenome, metabolome, host transcriptome and proteome from 99 patients with COPD and 36 healthy individuals in China. Multi-omics data were integrated using sequential mediation analysis, to assess in silico associations of the microbiome with two primary COPD inflammatory endotypes, neutrophilic or eosinophilic inflammation, mediated through microbial metabolic interaction with host gene expression. Hypotheses of microbiome-metabolite-host interaction were identified by leveraging microbial genetic information and established metabolite-human gene pairs. A prominent hypothesis for neutrophil-predominant COPD was altered tryptophan metabolism in airway lactobacilli associated with reduced indole-3-acetic acid (IAA), which was in turn linked to perturbed host interleukin-22 signalling and epithelial cell apoptosis pathways. In vivo and in vitro studies showed that airway microbiome-derived IAA mitigates neutrophilic inflammation, apoptosis, emphysema and lung function decline, via macrophage-epithelial cell cross-talk mediated by interleukin-22. Intranasal inoculation of two airway lactobacilli restored IAA and recapitulated its protective effects in mice. These findings provide the rationale for therapeutically targeting microbe-host interaction in COPD.

Inflammatory Endotype-Associated Airway Resistome in Chronic Obstructive Pulmonary Disease.

Antimicrobial resistance is a global concern in chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD). The collection of antibiotic resistance genes or resistome in human airways may underlie the resistance. COPD is heterogeneous, and understanding the airway resistome in relation to patient phenotype and endotype may inform precision antibiotic therapy. Here, we characterized the airway resistome for 94 COPD participants at stable disease. Among all demographic and clinical factors, patient inflammatory endotype was associated with the airway resistome. There were distinct resistome profiles between patients with neutrophilic or eosinophilic inflammation, two primary inflammatory endotypes in COPD. For neutrophil-predominant COPD, the resistome was dominated by multidrug resistance genes. For eosinophil-predominant COPD, the resistome was diverse, with an increased portion of patients showing a macrolide-high resistome. The differential antimicrobial resistance pattern was validated by sputum culture and in vitro antimicrobial susceptibility testing. Ralstonia and Pseudomonas were the top contributors to the neutrophil-associated resistome, whereas Campylobacter and Aggregatibacter contributed most to the eosinophil-associated resistome. Multiomic analyses revealed specific host pathways and inflammatory mediators associated with the resistome. The arachidonic acid metabolic pathway and matrix metallopeptidase 8 (MMP-8) exhibited the strongest associations with the neutrophil-associated resistome, whereas the eosinophil chemotaxis pathway and interleukin-13 (IL-13) showed the greatest associations with the eosinophil-associated resistome. These results highlight a previously unrecognized link between inflammation and the airway resistome and suggest the need for considering patient inflammatory subtype in decision-making about antibiotic use in COPD and broader chronic respiratory diseases. IMPORTANCE Antibiotics are commonly prescribed for both acute and long-term prophylactic treatment in chronic airway disorders, such as chronic obstructive pulmonary disease (COPD), and the rapid growth of antibiotic resistance is alarming globally. The airway harbors a diverse collection of microorganisms known as microbiota, which serve as a reservoir for antibiotic resistance genes or the resistome. A comprehensive understanding of the airway resistome in relation to patient clinical and biological factors may help inform decisions to select appropriate antibiotics for clinical therapies. By deep multiomic profiling and in vitro phenotypic testing, we showed that inflammatory endotype, the underlying pattern of airway inflammation, was most strongly associated with the airway resistome in COPD patients. There were distinct resistome profiles between neutrophil-predominant and eosinophil-predominant COPD that were associated with different bacterial species, host pathways, and inflammatory markers, highlighting the need of considering patient inflammatory status in COPD antibiotic management.

Dissociation between airway and systemic autoantibody responses in chronic obstructive pulmonary disease.

BACKGROUND: Autoimmune processes have been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the relationship between airway and systemic autoantibody responses remains unclear. The aim of this study was to elucidate this relationship in patients with stable COPD by investigating the correlation patterns between sputum and serum autoantibodies. METHODS: In this cross-sectional study, sputum supernatant and serum obtained from 47 patients with stable COPD were assayed for the presence of IgG antibodies against ten autoantigens: Smith antigen (Sm), ribosomal phosphoprotein P0 (P0), Ro/Sjögren syndrome type A antigen (Ro/SSA), La/Sjögren syndrome type B antigen (La/SSB), DNA topoisomerase I (Scl-70), histidyl-tRNA synthetase (Jo-1), U1 small nuclear ribonucleoprotein (U1-SnRNP), thyroid peroxidase (TPO), proteinase-3 (PR3), and myeloperoxidase (MPO). A second cohort of 55 stable COPD patients was recruited for validation, and a group of 59 non-COPD controls and a group of 20 connective-tissue disease-associated interstitial lung disease (CTD-ILD) patients were also recruited for comparison. Hierarchical clustering and network analysis were used to evaluate the correlation patterns between sputum and serum autoantibody profiles. RESULTS: Both hierarchical clustering and network analysis showed that sputum and serum autoantibody profiles were distinct in either analytic COPD cohort or validation cohort. In contrast, the autoantibodies of the two compartments in non-COPD controls and CTD-ILD patients were inadequately distinguished using either hierarchical clustering or network analysis. Many autoantibodies in the sputum were found to have significant correlations with lung function, symptom score and frequency of prior exacerbations in COPD patients, but the antibodies in the serum were not. CONCLUSIONS: We observed a dissociation between sputum autoantibodies and serum autoantibodies in patients with stable COPD, suggesting that airway and systemic immune status may play very different roles in the disease. Sputum autoantibodies are more clinically relevant than serum autoantibodies. Focusing on airway autoimmunity may help improve understanding of the immunopathological mechanism of COPD.

Sputum and serum autoantibody profiles and their clinical correlation patterns in COPD patients with and without eosinophilic airway inflammation.

BACKGROUND: Autoimmunity plays a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the autoantibody responses and their clinical correlation patterns in COPD patients with and without airway eosinophilic inflammation are unknown. The aim of this study was to compare the autoantibody profiles and their clinical associations in stable COPD patients, stratified by airway inflammatory phenotypes. METHODS: Matched sputum and serum, obtained from 62 stable COPD patients and 14 age-matched controls, were assayed for the presence of IgG and IgM antibodies against 13 autoantigens using protein array. A sputum eosinophil count ≥3% was used as cut-off value to stratify COPD patients into eosinophilic and non-eosinophilic groups. Correlation network analysis was used to evaluate the correlation patterns among autoantibody and clinical variables in each group. RESULTS: There were no significant differences of clinical parameters and autoantibody levels between the two COPD groups. In non-eosinophilic COPD, sputum anti-CytochromeC_IgG and anti-Aggrecan_IgM were significantly higher than those in healthy controls, and prior exacerbation was positively associated with lung function and sputum anti-Collagen-IV_IgG. While in eosinophilic COPD, sputum/serum anti-heat shock protein (HSP)47_IgG, serum anti-HSP70_IgG and serum anti-Amyloid-beta_IgG were significantly lower than those in healthy controls, and no significant correlation between prior exacerbations and lung function was found. Differences were also observed in network hubs, with the network for non-eosinophilic COPD possessing 9 hubs comprising two lung function parameters and seven autoantibodies, compared with eosinophilic COPD possessing 12 hubs all comprising autoantibodies. CONCLUSIONS: Autoantibody responses were heterogeneous and differentially correlated with the exacerbation risk and other clinical parameters in COPD patients of different inflammatory phenotypes. These findings provide useful insight into the need for personalized management for preventing COPD exacerbations.

Reproducibility of fluid-phase measurements in PBS-treated sputum supernatant of healthy and stable COPD subjects.

Purpose: The purpose of this study was to investigate the reproducibility of fluid-phase measurements in PBS-treated sputum supernatant, processed using the two-step method, of healthy and stable COPD individuals. Methods: Nine healthy subjects and 23 stable COPD patients provided sputum twice within 6 days. A two-step sputum processing method was used to obtain PBS-treated supernatant and sputum cells. Soluble protein markers and IgG and IgM autoantibody profiles in PBS supernatant were analyzed using customized microarrays. Repeatability of measurements was assessed by paired-sample testing and an intraclass correlation coefficient, then graphically reported by Bland-Altman plot. Results: There was no significant difference between the repeated detection of 8/10 types of soluble protein markers, all 13 types of IgG autoantibodies, and 12/13 types of corresponding IgM autoantibodies in PBS supernatant. The repeatability of measurements in PBS supernatant was substantial to very good for interleukin 6 (IL6), IL8, IL13, IL10, IL33, vascular endothelial growth factor, soluble receptor for advanced glycation end-products, and tumor necrosis factor-α; for IgG autoantibodies against aggrecan, centromere protein B (CENP-B), collagen II, collagen IV, cytochrome C, elastin, heat shock protein 47 (HSP47), HSP70, and La/Sjögren syndrome type B antigen; for IgM autoantibodies against CENP-B, collagen I, collagen II, collagen IV, cytokeratin 18, and HSP70; and for sputum neutrophils, macrophages and eosinophils count. Bland-Altman plots suggested good consistency within repeated measurements. Stable COPD patients differed from healthy subjects in the proportion of neutrophils and eosinophils; relative fluorescence intensity of anti-cytochrome C IgG, anti-aggrecan IgM, and anti-cytochrome C IgM. There was a significant positive correlation for stable COPD patients between sputum anti-collagen II IgG and post-bronchodilator FEV1%. Conclusion: We confirmed fluid-phase measurements in PBS-treated sputum supernatant by high-throughput techniques with good repeatability. We demonstrated the presence of IgG and IgM autoantibodies to multiple antigens in the airways of COPD patients.

[Epidemiological survey and analysis of asthma in children aged 0-14 years old in urban and rural areas of Chengdu region].

OBJECTIVE: To investigate the prevalence and incidence of asthma among children aged 0-14 years, with different living environments, economic levels, and sanitary conditions, in the urban and rural areas of Chengdu, China, and their influential factors. METHODS: Children aged 0-14 years who were selected from urban, suburban and rural areas of Chengdu, were included in the study. The subjects were selected from all children aged 0-14 years in schools, kindergartens and communities by random, cluster and non-proportional sampling. Parents were surveyed by questionnaire to find out suspected cases, which were then confirmed by inquiry and physical examination in the department of respiratory medicine. All the obtained data were analyzed using SPSS/PC statistical software. RESULTS: A total of 12082 children from the urban areas, 5677 from suburban areas and 5590 from the rural areas were included in the study. Of all the subjects, 551 (4.56%) had confirmed asthma, 150 (2.64%) had cough variant asthma (CVA), and 142 (2.54%) had suspected asthma. The prevalence rate of asthma was significantly higher in the urban areas than in the suburban and rural areas. The correct rate of diagnosis of asthma and CVA was highest in the urban areas, followed by the suburban and rural areas. Use of antibiotics and systemic corticosteroids was most common in the rural areas, followed by the suburban and urban areas, but this pattern was reversed for use of inhaled corticosteroids and leukotriene modifier. All the results in the three areas demonstrated that sex, age, age at which the first attack occurred, respiratory tract infection, inhalation/intake of allergens and genetic factors were significantly associated with asthmatic attack. CONCLUSIONS: Population density, living environment, medical and health resources and economic level are associated with the prevalence and treatment of asthma.

Epidemiological survey and analysis of asthma in children aged 0-14 years old in urban and rural areas of Chengdu region.

OBJECTIVES: To investigate the prevalence and incidence of asthma among children aged 0-14 years, with different living environments, economic levels, and sanitary conditions, in the urban and rural areas of Chengdu, China, and their influential factors. METHODS: Children aged 0-14 years who were selected from urban, suburban, and rural areas of Chengdu were included in the study. The subjects were selected from all children aged 0-14 years in schools, kindergartens, and communities by random, cluster and non-proportional sampling. Parents were surveyed by questionnaire to find out suspected cases, which were then confirmed by inquiry and physical examination in the departments of respiratory medicine. All the obtained data were analyzed using SPSS statistical software. RESULTS: A total of 12,082 children from the urban areas, 5,677 from suburban areas, and 5,590 from the rural areas were included in the study. Of all the subjects, 551 (4.56%) had confirmed asthma, 150 (2.64%) had cough variant asthma (CVA), and 142 (2.54%) had suspected asthma. The prevalence rate of asthma was significantly higher in the urban areas than in the suburban and rural areas. The correct rate of diagnosis of asthma and CVA was highest in the urban areas, followed by the suburban and rural areas. Use of antibiotics and systemic corticosteroids was most common in the rural areas, followed by the suburban and urban areas, but this pattern was reversed for use of inhaled corticosteroids and leukotriene modifier. All the results in the three areas demonstrated that sex, age, age at which the first attack occurred, respiratory tract infection, inhalation/intake of allergens, and genetic factors were significantly associated with asthmatic attack. CONCLUSIONS: Population density, living environment, medical and health resources and economic level are associated with the prevalence and treatment of asthma.