Airway Aging and Methylation Disruptions in HIV-associated Chronic Obstructive Pulmonary Disease

Abstract

Rationale: Age-related diseases like chronic obstructive pulmonary disease (COPD) occur at higher rates in people living with human immunodeficiency virus (PLWH) than in uninfected populations. Objectives: To identify whether accelerated aging can be observed in the airways of PLWH with COPD, manifest by a unique DNA methylation signature. Methods: Bronchial epithelial brushings from PLWH with and without COPD and HIV-uninfected adults with and without COPD (N = 76) were profiled for DNA methylation and gene expression. We evaluated global Alu and LINE-1 methylation and calculated the epigenetic age using the Horvath clock and the methylation telomere length estimator. To identify genome-wide differential DNA methylation and gene expression associated with HIV and COPD, robust linear models were used followed by an expression quantitative trait methylation (eQTM) analysis. Measurements and Main Results: Epigenetic age acceleration and shorter methylation estimates of telomere length were found in PLWH with COPD compared with PLWH without COPD and uninfected patients with and without COPD. Global hypomethylation was identified in PLWH. We identified 7,970 cytosine bases located next to a guanine base (CpG sites), 293 genes, and 9 expression quantitative trait methylation-gene pairs associated with the interaction between HIV and COPD. Actin binding LIM protein family member 3 (ABLIM3) was one of the novel candidate genes for HIV-associated COPD highlighted by our analysis. Conclusions: Methylation age acceleration is observed in the airway epithelium of PLWH with COPD, a process that may be responsible for the heightened risk of COPD in this population. Their distinct methylation profile, differing from that observed in patients with COPD alone, suggests a unique pathogenesis to HIV-associated COPD. The associations warrant further investigation to establish causality.

Keywords: COPD; HIV; airway epithelium; epigenomics; gene expression.

Figure 1.

Study overview and analysis pipeline. Bronchial epithelial brushings from four groups (HIV+/chronic obstructive pulmonary disease [COPD]+, HIV+/COPD−, HIV−/COPD+, HIV−/COPD−) (N = 76) were profiled for DNA methylation and gene expression. Quality controls and processing steps were applied to the DNA methylation and RNA sequencing data. The DNA methylation profile was used to explore epigenetic age and global methylation changes associated with HIV and COPD. Robust linear model equations were used to identify: 1) differential methylated cytosine bases located next to a guanine base (CpGs) and differentially expressed genes; and 2) expression quantitative trait methylation sites. Significant associations were defined at a false discovery rate < 0.1. This figure was created with BioRender.com. CPM = counts per million.

Figure 2.

Age acceleration and global methylation changes in HIV and chronic obstructive pulmonary disease (COPD). (A and B) Age acceleration and telomere shortening, respectively, based on HIV and COPD status. The y-axes on the plots represent the residuals obtained from the regression of methylation age (A) or telomere length estimator (B) on the chronological age. Significant P values (P < 0.05) shown inside the plots correspond to the Tukey’s honestly significant difference adjusted P value for the comparison between the groups.

Figure 3.

Differential DNA methylation and gene expression in the airway epithelium of patients with HIV and chronic obstructive pulmonary disease (COPD). (A and B) Differentially methylated positions (DMPs). (C and D) Differentially expressed genes (DEGs). The x-axes on A and C (HIV), and B and D (COPD), represent the effect size difference of the DMPs (β difference) and DEGs (log₂ fold change [FC] in gene expression). The y-axes represent the level of statistical significance for each DMP or DEG. The red and blue colors represent hypomethylation and hypermethylation for DMPs (A and B), respectively. The red and blue colors represent downregulation and upregulation for DEGs (C and D), respectively. Reference group for the A and C are non-HIV, and non-COPD is the reference group for B and D. The dotted horizontal line in each panel represents the −log₁₀ P value that corresponds to the false discovery rate < 0.10. The total number of DMPs (A and B) and DEGs (C and D) is shown at the top right corner of each panel.

Figure 4.

Airway epithelium cis–expression quantitative trait methylations (cis-eQTMs) identified in patients with HIV and chronic obstructive pulmonary disease (COPD). The figure illustrates the differential methylation and gene expression status for eQTMs (false discovery rate [FDR] < 0.1) in the airway epithelium. (A) Overlap between cis-eQTMs and HIV differentially methylated positions (DMPs) and expressed genes (DEGs). (B) Overlap between cis-eQTMs and COPD DMPs and DEGs. (C) Overlap between cis-eQTMs and disease interaction (HIV by COPD) DMPs and DEGs. The x-axes represent the level of statistical significance for the differential methylation of the cis-eQTMs multiplied by the direction of their effect. The y-axes represent the level of statistical significance for the differential gene expression of each cis-eQTM corresponding gene (multiplied by the direction of their effect size). The dotted horizontal and vertical lines represent the −log₁₀ P value that corresponds to the FDR < 0.1 for differential DNA methylation and gene expression.