DNA Methylation Changes in Lung Immune Cells Are Associated with Granulomatous Lung Disease

Abstract

Epigenetic marks are likely to explain variability of response to antigen in granulomatous lung disease. The objective of this study was to identify DNA methylation and gene expression changes associated with chronic beryllium disease (CBD) and sarcoidosis in lung cells obtained by BAL. BAL cells from CBD (n = 8), beryllium-sensitized (n = 8), sarcoidosis (n = 8), and additional progressive sarcoidosis (n = 9) and remitting (n = 15) sarcoidosis were profiled on the Illumina 450k methylation and Affymetrix/Agilent gene expression microarrays. Statistical analyses were performed to identify DNA methylation and gene expression changes associated with CBD, sarcoidosis, and disease progression in sarcoidosis. DNA methylation array findings were validated by pyrosequencing. We identified 52,860 significant (P < 0.005 and q < 0.05) CpGs associated with CBD; 2,726 CpGs near 1,944 unique genes have greater than 25% methylation change. A total of 69% of differentially methylated genes are significantly (q < 0.05) differentially expressed in CBD, with many canonical inverse relationships of methylation and expression in genes critical to T-helper cell type 1 differentiation, chemokines and their receptors, and other genes involved in immunity. Testing of these CBD-associated CpGs in sarcoidosis reveals that methylation changes only approach significance, but are methylated in the same direction, suggesting similarities between the two diseases with more heterogeneity in sarcoidosis that limits power with the current sample size. Analysis of progressive versus remitting sarcoidosis identified 15,215 CpGs (P < 0.005 and q < 0.05), but only 801 of them have greater than 5% methylation change, demonstrating that DNA methylation marks of disease progression changes are more subtle. Our study highlights the significance of epigenetic marks in lung immune response in granulomatous lung disease.

Keywords: berylliosis; epigenetics; genomics; immunology; sarcoidosis.

Figure 1.

Differentially methylated single-CpG probes in BAL cells associated with chronic beryllium disease (CBD). (A) A volcano plot of false discovery rate (FDR) adjusted q values for disease status (CBD/beryllium-sensitized [BeS]) from the ANOVA model plotted as a function of methylation change (CBD − BeS). Probes with statistically significant change q < 0.05 and methylation change >25% are highlighted in red. (B) Protein–protein interactome analysis of 1,944 genes with significant CBD-associated DNA methylation changes (q < 0.05, P < 0.005, and methylation change >25%). The zero-order interactome was created using NetworkAnalyst (57) and the STRING PPI dataset. The nodes are colored based on their methylation (green are hypomethylated, whereas red are hypermethylated). The sizes of nodes are proportional to their betweenness centrality values. (C) Methylation-expression plot for genes with CBD-associated DNA methylation changes with q < 0.05, P < 0.005; x-axis methylation difference is represented by the mean % methylation difference in CBD compared with BeS subjects; y-axis expression difference is represented by the mean fold change in CBD compared with subjects with BeS. The black solid circles represent hypomethylated genes that were associated with increased gene expression as well as some hypermethylated genes associated with decreased gene expression. The red solid circles represent methylation changes that were not associated with canonical gene expression differences.

Figure 2.

DNA methylation and gene expression are more variable in sarcoidosis (Sarc) than CBD. Representative plots of (A) DNA methylation and (B) relationship of DNA methylation with gene expression of genes from Figure 1; BeS, red; CBD, blue; sarcoidosis, green.

Figure 2.

DNA methylation and gene expression are more variable in sarcoidosis (Sarc) than CBD. Representative plots of (A) DNA methylation and (B) relationship of DNA methylation with gene expression of genes from Figure 1; BeS, red; CBD, blue; sarcoidosis, green.

Figure 3.

Differentially methylated single-CpG probes in BAL cells associated with disease progression in sarcoidosis. (A) A volcano plot of FDR-adjusted q values for disease status (progressive/remitting sarcoidosis) from the ANOVA model plotted as a function of methylation change (progressive − remitting sarcoidosis). Probes with statistically significant change q < 0.05 and methylation change >5% are highlighted in red. (B) Methylation-expression plot for genes with progressive sarcoidosis-associated DNA methylation changes with q < 0.05; x-axis methylation difference is represented by the mean % methylation difference in progressive compared with remitting sarcoidosis subjects; y-axis expression difference is represented by the mean fold change in progressive compared with remitting sarcoidosis subjects. The black solid circles represent hypomethylated genes that were associated with increased gene expression as well as some hypermethylated genes associated with decreased gene expression. The red solid circles represent methylation changes that were not associated with canonical gene expression differences.