Bleecker, Eugene R.

Introduction Despite the identification of multiple susceptibility loci by genome-wide association studies (GWAS), considerable chronic obstructive pulmonary disease (COPD) heritability remains unexplained. Aim To identify interaction networks of airway epithelial cell DNA methylation in COPD and further explore potential correlations with airway bacterial composition, as potentially collective regulators of biological pathways influencing COPD severity. Methods Using DNA isolated from bronchial airway brushings of 67 ever-smokers (>20 pack-years) from the SubPopulations and InteRmediate Outcomes Measures in COPD Study (SPIROMICS), we assessed proportion of DNA methylation (β) by epigenome-wide association study (EWAS) and examined associations of differentially methylated CpG probes (DMPs) with risk for moderate-to-severe COPD (N=34) versus absent or mild COPD (N=33). We tested co-methylation modules generated by Weighted Correlation Network Analyses (WGCNA) for associations with moderate-to-severe COPD and with bacterial genus-level relative abundances (16S rRNA sequencing). Results EWAS-identified nominally significant DMPs enriched for lung function GWAS loci. Eigengenes in six WGCNA modules were associated with moderate-to-severe COPD (false discovery rate <0.05). Four of those modules were enriched for forced expiratory volume in 1 s/forced vital capacity GWAS loci, and five overlapped with DMPs from EWAS. Overlapping CpG loci in three COPD-associated modules were adjacent to mucin genes; one had 10 genes highly ranked by connectivity with MUC5B, including important pathway genes: B3GNT6 , DGKI and ITGA8 . CpGs in an independent COPD-associated module showed the most correlations with Rothia , with directionality suggestive of negative associations with moderate-severe COPD. Conclusions Bronchial epithelial DNA methylation modules enriched for lung function GWAS loci associate with COPD severity in SPIROMICS. Potential module relationships to bronchial bacterial composition require further validation.

Abstract Rationale Inflammation is central to chronic obstructive pulmonary disease (COPD) pathogenesis but incompletely represented in COPD prognostic models. The neutrophil-to-lymphocyte ratio (NLR) is a readily available inflammatory biomarker. Objectives To explore the associations of NLR with smoking status, clinical features of COPD, and future adverse outcomes. Methods We analyzed NLR calculated from the complete blood count of participants who currently or formerly smoked (n = 2,624) and tobacco-naive control subjects (n = 187) in the SPIROMICS multicenter observational cohort study. We assessed the stability of NLR at 6 weeks and 1 year, the association with select blood biomarkers, and the impact of smoking on NLR and cell counts. We stratified participants by NLR quartiles to compare cross-sectional clinical features at enrollment, prospectively observed exacerbations at 1 year, and mortality during longitudinal follow up. Results Higher NLR quartiles were broadly associated with more severe clinical features of COPD. NLR values were repeatable at 6 weeks (intraclass correlation coefficient, 0.74) and 1 year (intraclass correlation coefficient, 0.62). The impact of smoking on NLR varied with the severity of airflow limitation, mediated by an interaction between smoking, forced expiratory volume in 1 second percent predicted, and neutrophil counts but not lymphocyte counts. The highest NLR quartile (&gt;3.11) was associated with an increased risk of exacerbation over 1 year (adjusted odds ratio, 1.51; 95% confidence interval, 1.18, 1.92) and increased risk of mortality (adjusted hazard ratio, 1.41; 95% confidence interval, 1.20, 1.66) compared with quartiles 1–3. Conclusions Elevated NLR in stable COPD is a widely available biomarker associated with increased risk for exacerbation and death. The impact of cigarette smoking on NLR varies with disease severity.

Rationale Immunoglobulin A (IgA) deficiency, a rare, highly heritable trait, is associated with frequent pulmonary infections, emphysema, airway changes and low lung function; however, it is unclear if reduced IgA levels may affect lung structure and function. Methods Serum IgA, IgA1 and galactose-deficient IgA1 (Gd-IgA1) levels were measured in the population-based Multi-Ethnic Study on Atherosclerosis (MESA). The MESA Lung Study measured percent emphysema on cardiac CT and airway dimensions on chest CT, and performed spirometry. Regression models were evaluated after adjustment for demographic and CT factors. Mendelian randomisation (MR) analyses were conducted using genetic variants from the Trans-Omics for Precision Medicine (TOPMed) programme. A replication analysis was performed in the SubPopulations and InteRmediate Outcome Measures In COPD Study (SPIROMICS). Measurements and main results Among 5497 participants, lower log-normalised serum IgA levels were associated with greater percent emphysema (β=−0.084; 95% CI −0.14 to –0.026; p=0.005), which was confirmed on MR (β=−0.79; 95% CI −1.4 to –0.18; p=0.011). Greater log-normalised serum Gd-IgA1 levels were associated with airway wall thickness (β=0.0079; 95% CI 0.0017 to 0.014; p=0.012; n=2580) and decline in the forced expiratory volume in one second (FEV1) (β=−0.012; 95% CI −0.021 to –0.0036; p=0.0055; n=2778) and FEV1/forced vital capacity (FVC) ratio (β=−0.0028; 95% CI −0.0048 to –0.00084; p=0.0054; n=2778). Conclusion Lower serum IgA levels were associated with greater percent emphysema. Additionally, higher Gd-IgA1 levels were associated with airway wall thickness and lung function decline. These findings support a protective role of IgA in emphysema pathogenesis and possible deleterious role of Gd-IgA1 in airway diseases.

Abstract Introduction: The airway inflammation of asthma is characterized by immune phenotypes, often classified as Type 2 inflammation high or low. Social determinants of health (SDoH) can influence asthma risk and severity, but whether or not SDoH influence immune phenotypes is unclear. The social deprivation index (SDI) is a composite measure of seven neighborhood level characteristics: poverty level, less than 12 years of education, single parent household, living in rented housing unit, living in overcrowded housing unit, households without a car, and non-employed adults under 65 years of age. The scale of SDI ranges from 1 to 100, correlating with increasing deprivation. We hypothesized that social deprivation influences immune phenotype of asthma, and that deprivation effects vary by sex and race. Methods: Using the American Community Survey summary file spanning 2015 to 2019, we linked SDI to participant zip codes for 680 Black and White participants in the Severe Asthma Research Program (SARP), a NHLBI-multicenter network phenotyping asthma across the US from 2001-2024 (table 1). We compared SDI among the cohort by sex, race and asthma severity, and correlated SDI to superoxide dismutase (SOD) and T2-high biomarkers, such as eosinophils, IgE and Fraction of Exhaled Nitric Oxide (FENO). Results: SDI is similar across healthy and asthmatic participants (SDI: median [interquartile range]: healthy: 40 [56]; asthmatics: 51 [53], P = 0.53). When evaluated by race, SDI is significantly higher in Black asthmatics, independent of sex (SDI: Black male: 76 [4], White male: 35 [45]; P&lt;0.0001; Black females: 77 [35], White females: 40 [45]; P&lt;0.00010). Within SARP asthmatics, SDI shows a positive association with the T2 inflammatory markers, log IgE (R=0.13 [90%CI: 0.1, 0.2)] and log FENO (R=0.15 [90%CI: 0.1, 0.2]), while it correlates negatively with log of absolute neutrophil count (R= -0.12 [90%CI: -0.19,-0.1]). The antioxidant enzyme SOD increases with SDI (R=0.11, [90% CI: 0.04, 0.17]). Notably, within Black asthmatic males only, SDI is associated with Type 2 biomarkers, showing a positive correlation with log FENO (R=0.24 [90%CI: 0.1,0.4]) and negative correlation with log IgE levels (R= -0.25, [90% CI: -0.4, -0.1]). No association was found between SDI and eosinophils or with SOD in Black males. Conclusions: Neighborhood level social deprivation is associated with a unique asthma phenotype in Black males. Black males living at high social deprivation have a lower IgE and higher FENO, with no change in SOD activity.

Abstract Rationale: Individuals with chronic obstructive pulmonary disease (COPD) have different clinical trajectories, some with minimal symptom burden, others experiencing recurrent exacerbations, the most severe of which require hospitalization. Existing diagnostics, including spirometry and symptom assessments, lack the precision to identify high-risk patients who may benefit from timely preventive therapies. Nuclear cell-free DNA (ncfDNA) is a well-established marker of systemic tissue injury, implicated in amplifying inflammation by triggering type 1 interferon release by activating DNA-sensing pathways. Because COPD is accompanied by inflammation and tissue injury systemically, we hypothesized that ncfDNA is associated with increased risk of severe exacerbations requiring hospitalization. Methods: Design: We utilized baseline plasma samples randomly selected from 672 individuals with COPD from the Subpopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS) cohort, which prospectively collected exacerbation numbers and type. The primary endpoint was exacerbation requiring hospitalization. Secondary endpoints included total number of exacerbations (defined as use of oral steroids, antibiotics, or healthcare utilization) and severe exacerbations (defined as requiring emergency room care or hospitalization). Measure: We measured plasma ncfDNA using digital droplet PCR. Analysis: We assessed the association of ncfDNA with exacerbation outcomes using negative binomial regression or Cox regression, adjusting for age, percent post-bronchodilator forced expiratory volume in one second (FEV1), supplemental oxygen use, and smoking status. Results: In total, 57% (n=383) of COPD participants experienced exacerbations, 29% (n=197) at least one severe, and 22% (n=145) requiring hospitalization, the latter at a median of 622 days from enrollment (IQR=287-997). Higher ncfDNA levels at enrollment correlated with measures of COPD severity, including baseline FEV1 (beta = -0.12, 95% CI -0.18 to -0.049, p &lt;0.001). Higher ncfDNA associated with increased risk of COPD exacerbation requiring hospitalization (incidence rate ratio (IRR) 1.40, 95% CI 1.12-1.75, p =0.003) and severe exacerbations (IRR 1.36, 95% CI 1.11-1.67, p=0.002) and a trend towards total exacerbations (IRR 1.11, 95% CI 0.98-1.26, p= 0.09). The primary outcome, risk of time-to-first hospitalization, correlated significantly with higher ncfDNA with hazard ratio (HR) 1.23, 95% CI 1.01-1.49, p=0.037. Conclusion: These results suggest that ncfDNA may serve as a predictor for severe and hospitalization-associated COPD exacerbations. To better understand the relationship between ncfDNA and COPD pathophysiology, follow-up studies should explore longitudinal trends and cfDNA tissue sources, and their link to COPD exacerbations.

Abstract Rationale. Bronchoscopy can directly assess cells in airway epithelium in participants with COPD, but it is invasive, expensive, difficult to perform at scale, and contraindicated in severe disease. Blood is much more easily available but contains only a limited subset of cell types, many of which are less relevant to lung biology. Recent advances in RNA-sequencing technology have allowed for reliable measurement of transcriptomics in a wider variety of more accessible tissues, including hair follicle cells. We collected and performed bulk RNA-seq on hair follicles as an ancillary study within the NIH-funded SubPopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) and investigated their cell type composition to better understand the opportunities and limitations of this tissue for the study of lung disease. Methods. SPIROMICS recruited individuals with and without COPD aged 40-80 years with ≥ 20 pack-years smoking history. Participant at several sites underwent collection of approximately 10 hair follicles from the head, which were immediately frozen at -80ºC in 2018-21. mRNA in hair follicles was measured by bulk RNA-seq, and cell type deconvolution was performed using GEDIT v1.7 with the provided BlueCodeV1.0.tsv reference matrix. Cell types were collapsed into broader categories by summing the estimated cell type proportions. Results. The cell type deconvolution of the hair follicle bulk RNA-seq from 80 participants with and without COPD estimated that the tissue was largely composed of immune cells (median 36%, including B cells, T cells, neutrophils, macrophages, NK cells, and monocytes) and epithelial cell types (median 31%), with the largest subgroup of epithelial cell types identified as hair follicular keratinocytes (Figure 1). The next largest component was fibroblasts (median 9%), followed by endothelial cells (median 8%). Signals for myocytes (median 5%), melanocytes (median 5%), and chondrocytes (median 5%) were also present. Conclusion. This initial study demonstrated promising signals for a variety of lung-relevant cell types in hair follicles in SPIROMICS, including immune and epithelial cells in addition to fibroblasts and endothelial cells that are not otherwise easily accessible. Hair follicle gene expression profiling shows promise for understanding numerous facets of lung biology and disease that both complements and exceeds that of other noninvasive tissues such as blood.

Abstract Asthma is a heterogeneous disease with variable presentation and characteristics. There is a critical need to identify underlying molecular endotypes of asthma. We performed the largest transcriptomic analysis of 808 bronchial epithelial cell samples across 11 independent cohorts, including 3 cohorts from the Severe Asthma Research Program. Using seven datasets (218 patients with asthma, 148 healthy control subjects) as discovery cohorts, we identified 505 differentially expressed genes, which we validated in the remaining four datasets. Unsupervised clustering using the 505 differentially expressed genes identified four reproducible clusters of patients with asthma across all datasets, corresponding to healthy control subjects, patients with mild/moderate asthma, and patients with severe asthma with significant differences in several clinical markers of severity, including pulmonary function, Type 2 inflammation, fractional exhaled nitric oxide, and maximum bronchodilator reversibility. Importantly, we found the same clusters in pediatric patients using nasal lavage fluid cells, demonstrating the gene signature and clusters are not confounded by age and are conserved in both lower and upper airways. The four asthma clusters may represent a unifying framework for understanding the molecular heterogeneity of asthma. Further study could potentially enable a precision medicine approach of matching therapies with patients with asthma most likely to benefit.

Abstract Rationale Among tobacco-exposed persons with preserved spirometry (TEPSs), we previously demonstrated that different lung volume indices—specifically, elevated total lung capacity (TLC) versus elevated ratio of functional residual capacity to TLC (FRC/TLC)—identify different lung disease characteristics in the COPDGene cohort. Objective We sought to determine differential disease characteristics and trajectories associated with lung volume indices among TEPSs in the SPIROMICS cohort. Methods We categorized TEPSs (n = 814) by tertiles (low, intermediate, and high) of TLC or residual volume–to-TLC ratio (RV/TLC) derived from baseline computed tomography images and then examined clinical and spirometric disease trajectories in mutually exclusive categories of participants with high TLC without high RV/TLC ([TLC]high) versus high RV/TLC without high TLC ([RV/TLC]high). We examined differences in computed tomography–measured emphysema (Hounsfield units [HU] ⩽−950; parametric response mapping [PRM] of emphysema), air trapping (HU⩽−856; PRM of functional small airway disease; a disease probability measure for non-emphysematous gas trapping), airway geometry (the mean square root of wall area of a hypothetical airway with 10 mm internal perimeter), respiratory symptoms (on the modified Medical Research Council Dyspnea Scale; COPD Assessment Test [CAT]; St. George’s Respiratory Questionnaire [SGRQ]; and Short Form-12 [SF12]), and outcomes (annualized exacerbation rate) between the two categories at baseline and over follow-up time up to 8.5 years, using regression modeling adjusted for age, sex, height, weight, and smoking status (current vs. former smoker) and burden (pack-years). Results In TEPSs, the pattern of spirometric disease progression differed between participants with [TLC]high and those with [RV/TLC]high: There was increased forced vital capacity with stable forced expiratory volume in 1 second in participants with [TLC]high, versus unchanged forced vital capacity but nominally decreased forced expiratory volume in 1 second in those with [RV/TLC]high. Compared with participants with [TLC]high, TEPSs with [RV/TLC]high had less emphysema (by HU ⩽−950) but more airway disease (by HU ⩽−856; PRM of functional small airway disease; disease probability measure for gas trapping, and mean square root of wall area of a hypothetical airway with 10 mm internal perimeter), more respiratory symptoms (on the modified Medical Research Council Dyspnea Scale, CAT, SGRQ, and SF12), and more severe exacerbations at baseline. Over an average follow-up of 4.1 ± 2.4 years (range = 0.5–8.5 yr), TEPSs with [RV/TLC]high also had a higher likelihood of developing more severe spirometric disease (preserved ratio impaired spirometry or Global Initiative for Chronic Obstructive Lung Disease Classification 2) and worsening of their respiratory symptoms (on the CAT and SGRQ). Although the incidence rates of respiratory exacerbations, hospitalizations, and mortality were not significantly different between the two categories over the follow-up period, TEPSs with [RV/TLC]high were more likely to have been prescribed a respiratory inhaler at their last follow-up visit. Conclusions In these TEPSs from the SPIROMICS cohort, lung volume stratification by TLC versus RV/TLC identifies two pre-COPD phenotypes with distinct respiratory symptoms, radiographic features, and clinical trajectories. The characteristics of these pre-COPD phenotypes match those previously described in the COPDGene cohort using TLC versus FRC/TLC stratification.