Multiomics links global surfactant dysregulation with airflow obstruction and emphysema in COPD

Abstract

Rationale: Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms.

Methods: Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed.

Results: Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log₂ fold change (log₂FC) -2.0, -2.2, -1.5, -0.5, -0.7 and -0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= -0.56, r= -0.58, r= -0.45, r= -0.36, r= -0.44, r= -0.37, r= -0.40 and r= -0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= -0.55, r= -0.61, r= -0.48, r= -0.51, r= -0.41, r= -0.31 and r= -0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log₂FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85).

Conclusions: Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease.

FIGURE 1

Bronchoalveolar lavage lipidomic analysis showed reduced levels of phosphatidylcholine (PC) phospholipids, specifically PC 32:0, dipalmitoyl phosphatidylcholine (DPPC), as well as phosphatidylglycerol (PG) in COPD. a) Volcano plots of lipid abundance in healthy volunteer never-smokers (HV-NS) versus healthy volunteer ex-smokers (HV-ES) (left), HV-ES versus COPD subjects (middle) and HV-NS versus COPD subjects (right). The x-axis displays log₂(fold change) and the y-axis displays −log₁₀(adjusted p-value). The dashed horizontal line represents an adjusted p-value threshold of 0.05. DPPC is labelled. Lipid classes, including PC, PG, phosphatidylinositols (PI), phosphatidylethanolamines (PE) and triglycerides (TG) are coloured. b) Covariate-adjusted box plots showing the summed abundance of PC, PG and PI compared across COPD and HV-ES/HV-NS cohorts. c) Covariate-adjusted box plot showing the composition of the top three most abundant PC lipids. d) Covariate-adjusted box plots showing the composition of the top three most abundant PG lipids. For details regarding covariate adjustment see the supplementary methods. *: adjusted p<0.05; **: adjusted p<0.01; ns: not significant.

FIGURE 2

Correlation analysis showed correlation between bronchoalveolar lavage fluid phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositols (PI), surfactant protein A (SFTPA), surfactant protein B (SFTPB), surfactant protein D (SFTPD) and napsin A (NAPSA), and forced expiratory value in 1 s (FEV_1­) to forced vital capacity (FVC) ratio in COPD. The colour of each voxel of the heatmap represents the calculated Spearman's correlation coefficient between a COPD lung function parameter and the abundance of a surfactant protein, the abundance of a surfactant-associated protein or the summed abundance of a lipid category. The y-axis displays protein symbols or lipid abbreviations and the x-axis displays lung function parameters. CTSH: cathepsin H; ELANE: neutrophil elastase; MMP: matrix metalloproteinase; %LAA: % low-attenuation areas; T_LCO: transfer coefficient of the lung for carbon monoxide; E/I MLD: expiratory to inspiratory mean lung density. *: adjusted p<0.05; **: adjusted p<0.01.

FIGURE 3

Bronchoalveolar lavage fluid (BALF) proteomic analysis showed lower surfactant proteins and proteins involved in surfactant synthesis and secretion in COPD. a) Volcano plots of protein abundance in healthy volunteer non-smoking subjects (HV-NS) versus healthy volunteer ex-smoking subjects (HV-ES) (left), HV-ES versus COPD subjects (middle) and HV-NS versus COPD subjects (right). The x-axis displays log₂(fold change) and the y-axis displays −log₁₀(adjusted p-value). The dashed horizontal line represents an adjusted p-value threshold of 0.05. Proteins whose abundance is significantly altered in COPD compared to HV-ES and HV-NS donors (–log₁₀(adjusted p)>1.3) are labelled on the volcano plots. These include surfactant and surfactant-associated proteins surfactant protein A (SFTPA), surfactant protein B (SFTPB), surfactant protein D (SFTPD), cathepsin H (CTSH), napsin A (NAPSA), CD44, neutrophil elastase (ELANE) and matrix metalloproteinase (MMP) 9. b) Covariate-adjusted box plots showing the abundance of surfactant proteins SFTPA, SFTPB, SFTPD and NAPSA, CTSH, CD44, ELANE and MMP9 across COPD and HV-ES/HV-NS cohorts. For details regarding covariate adjustment see the supplementary methods. c) ELISA showing concentrations of BALF surfactant protein (SP)-D. ELISA was performed using a rabbit polyclonal anti-recombinant fragment of human SP-D capture antibody and biotinylated mouse anti-human SP-D detection antibody with streptavidin–horseradish peroxidase. Quantification was through comparison with a recombinant full-length human SP-D standard. *: adjusted p<0.05; **: adjusted p<0.01.

FIGURE 4

Serum proteomic analysis detected increased surfactant protein D (SFTPD) and surfactant protein B (SFTPB) in COPD patients. a) Box plot of serum SFTPD abundance in healthy non-smoking volunteer subjects (HV-NS), healthy ex-smoking volunteer subjects (HV-ES) and COPD subjects. The y-axis is the intensity corresponding to SFTPD for each donor where it was detected. b) Spearman's rank correlation of serum and donor-matched bronchoalveolar lavage fluid (BALF) SFTPD abundance across the cohort. c) Two-by-two contingency table for missing values in SFTPB in relation to disease status (i.e. COPD, HV-ES and HV-NS). p-values are obtained from Fisher's exact test, which compares the proportion of missing values in COPD and HV-ES/HV-NS cohorts. The left table compares COPD and HV-ES cohorts, whereas the right table compares COPD and HV-NS cohorts. d) Spearman's rank correlation of serum and donor-matched BAL cathepsin H (CTSH) abundance across cohort. e) receiver operator characteristics-curve of the logistic regression model trained on all donor-matched serum samples. AUC: area under the curve. *: p<0.05; **: p<0.01.