Lower Airway Dysbiosis Augments Lung Inflammatory Injury in Mild-to-Moderate Chronic Obstructive Pulmonary Disease

Abstract

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

Keywords: COPD; lung inflammation; metatranscriptomics; microbiome; transcriptomics.

Figure 1.

Lower airway dysbiotic signatures in mild-to-moderate chronic obstructive pulmonary disease as compared with smoker control subjects. Differential enrichment analysis based on edgeR (with multiple comparison adjustment based on false discovery rate [FDR]) on (A) 16S rRNA gene sequencing, displaying genera amplicon sequence variant; (B) whole-genome sequencing; and (C) RNA metatranscriptome sequencing. Only the top differentially enriched (based on fold change) and significant data with multiple comparisons based on FDR are displayed. Bubble size is based on relative abundance for each dataset separately. WGS = whole-genome sequencing.

Figure 2.

Changes in lower airway host transcriptome in chronic obstructive pulmonary disease (COPD). (A) PC analysis comparing subjects with COPD and smoker control (SC) subjects with permutational multivariate ANOVA P value. (B) Volcano plot showing differentially enriched genes based on fold change versus log10 adjusted P value (false discovery rate [FDR]) using edgeR comparing COPD and SC subjects. Genes with a log fold change >0 are enriched in COPD and <0 are enriched in SC. With an FDR cutoff of 0.2, only genes with an FDR < 0.2 are colored (green or red). (C) Ingenuity pathway analysis identifying cascade of upstream transcriptional regulators and associated disease or function relating to overlapping gene sets found using Gene Set Enrichment Analysis of our cohort and the publicly available GSE37147 dataset. PC = principal component.

Figure 3.

Effects of lower airway dysbiosis in a preclinical model of smoke-induced lower airway injury. (A) Murine model and exposures. (B) MLI calculated in (C) 20 different lung sections per mouse (P value, Mann-Whitney *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (D) Heat map of log-transformed cytokines or chemokines (pg/ml) normalized by grams of lung weight (n = 5 per group). Mann-Whitney comparisons demonstrate statistically significant comparisons (⁺phosphate-buffered saline [PBS] vs. smoke, P < 0.05; ^$smoke vs. mixture of oral commensals [MOC], P < 0.05; ^#MOC vs. MOC + smoke, P < 0.05; *PBS vs. MOC, P < 0.05; ^%smoke vs. MOC + smoke, P < 0.05; and ^@PBS vs. MOC + smoke, P < 0.05; n = 5 mice per group). MLI = mean linear intercept.

Figure 4.

Effects of lower airway dysbiosis in a preclinical model of smoke-induced lower airway inflammation. (A) Flow cytometry of single-cell suspensions of lung homogenates with differences between PBS, smoke, mixture of oral commensals (MOC), and MOC + smoke groups. (B) Differences in cell composition in the airways and parenchyma identified using immunohistochemistry (IHC) performed on the lung sections of five mice per group and obtained from 20 different sections per mouse (P value Mann-Whitney, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (C) Representative IHC images (DAPI = nuclei; orange = Mac; green = CD3⁺; yellow = CD4⁺; red = CD8⁺; and pink = Neu]). Mac = macrophage; Neut = neutrophils; PBS = phosphate-buffered saline.

Figure 5.

Effects of lower airway dysbiosis on the lung transcriptome. (A) PC analysis based on Bray-Curtis dissimilarity index on host lung transcriptome comparing all four exposures, permutational multivariate ANOVA P < 0.01. (B) Volcano plots showing differentially enriched genes [DEGs] identified by fold change versus log10 adjusted (false discovery rate) P value based on edgeR analyses between different exposures. (C) Gene Set Enrichment Analysis using DEGs identified significant and concordant pathways for experimental conditions in the preclinical mouse model with those identified in the human cohort (subjects with chronic obstructive pulmonary disease [COPD] vs. smoker control [SC] subjects). First comparing the mouse smoke versus phosphate-buffered saline (PBS) analysis to the human COPD versus SC analysis, then the mouse mixture of oral commensals (MOC) versus PBS analysis to the human COPD versus SC analysis, followed by the mouse MOC + smoke versus PBS analysis to the human COPD versus SC analysis, and finally the mouse MOC + smoke versus smoke analysis to the human COPD versus SC analysis. (D) Ingenuity pathway analysis comparing regulation of canonical pathways between different conditions in the preclinical mouse model and those identified in the human COPD versus SC cohort. The values for the heatmap represent a z-score. PC = principal component.