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Genome-wide DNA methylation profiling shows a distinct epigenetic signature associated with lung macrophages in cystic fibrosis - PubMed

  • ️Mon Jan 01 2018

Genome-wide DNA methylation profiling shows a distinct epigenetic signature associated with lung macrophages in cystic fibrosis

Youdinghuan Chen et al. Clin Epigenetics. 2018.

Abstract

Background: Lung macrophages are major participants in the pulmonary innate immune response. In the cystic fibrosis (CF) lung, the inability of lung macrophages to successfully regulate the exaggerated inflammatory response suggests dysfunctional innate immune cell function. In this study, we aim to gain insight into innate immune cell dysfunction in CF by investigating alterations in DNA methylation in bronchoalveolar lavage (BAL) cells, composed primarily of lung macrophages of CF subjects compared with healthy controls. All analyses were performed using primary alveolar macrophages from human subjects collected via bronchoalveolar lavage. Epigenome-wide DNA methylation was examined via Illumina MethylationEPIC (850 K) array. Targeted next-generation bisulfite sequencing was used to validate selected differentially methylated CpGs. Methylation-based sample classification was performed using the recursively partitioned mixture model (RPMM) and was tested against sample case-control status. Differentially methylated loci were identified by fitting linear models with adjustment of age, sex, estimated cell type proportions, and repeat measurement.

Results: RPMM class membership was significantly associated with the CF disease status (P = 0.026). One hundred nine CpG loci were differentially methylated in CF BAL cells (all FDR ≤ 0.1). The majority of differentially methylated loci in CF were hypo-methylated and found within non-promoter CpG islands as well as in putative enhancer regions and DNase hyper-sensitive regions.

Conclusions: These results support a hypothesis that epigenetic changes, specifically DNA methylation at a multitude of gene loci in lung macrophages, may participate, at least in part, in driving dysfunctional innate immune cells in the CF lung.

Keywords: Bronchoalveolar lavage; DNA methylation; Epigenetics; Lung macrophages.

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Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the Committee for the Protection of Human Subjects at the Geisel School of Medicine at Dartmouth (#22781). All study volunteers provided written informed consent for participation.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1

DNA methylation landscape in CF versus healthy controls. Recursively partitioned mixture model (RPMM) of the 10,000 CpGs (rows) with greatest sample variance across subjects. Individual samples 1–16 are shown in columns with sample status bar at the top: black (CF) and gray (healthy). Blue color represents increased sample methylation (a). RPMM determined the similarity of methylation among subjects resulting in two methylation classes L and R (b). Methylation class membership was associated with class status; inset contingency table depicts subject distribution in each class via a two-tailed Fisher’s exact test (P = 0.026)

Fig. 2
Fig. 2

Cellular composition and heterogeneity profiling. Heat map illustrates reference-free deconvolution (RefFreeEWAS) of putative cell type and size proportions across the n = 16 samples included in this study (a). Healthy subjects had higher proportions of putative cell type/size 1 (P = 0.014), and CF subjects had a higher proportion of cell type/size 2 (P = 0.018) (b)

Fig. 3
Fig. 3

Bronchoalveolar lavage cell cytospins. Bronchoalveolar lavage (BAL) samples were obtained from tertiary airways in the right upper lobe of subjects. BAL cells were isolated and prepared as cytospins as described in the “Methods” section. BAL cells from healthy subjects are a mostly homogeneous population of cells (lung macrophages (LM)) with oval to reniform nuclei and abundant cytosol (open arrow) (a). Cytospins of BAL cells from CF subjects (CD15-depleted) (b) show a majority population of LMs (open arrow) as well as smaller roundish cells with darker staining nuclei and less cytosol (blue arrowheads) and cells containing variably shaped and stained nuclei (black arrowheads). Images shown are representative of multiple subjects

Fig. 4
Fig. 4

Epigenome-wide differential methylation in CF. Comparative analysis of DNA methylation in CF and healthy subjects identified 109 differentially methylated CpGs (FDR P < 0.1, a). CpG hyper-methylated in CF compared to controls (green) and hypo-methylated CpGs (blue) are plotted as log2 fold increase or decrease in methylation M value (x-axis) versus log10 FDR-adjusted P value (y-axis). Statistically significant CpGs associated with specific genes are labeled, and unlabeled points represent CpGs associated with no known gene at that location. Enrichment of differentially methylated CpGs to genomic and transcriptional context is shown in forest plots (b), illustrating that hypo-methylated CpGs in CF BAL are enriched for enhancer regions and CpG islands and that hyper-methylated CpGs in CF BAL are under-represented for gene promoter regions

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