Epigenome profiling reveals significant DNA demethylation of interferon signature genes in lupus neutrophils

Abstract

Recent evidence suggests that neutrophils play an important role in the pathogenesis of lupus. The goal of this study was to characterize the epigenetic architecture, by studying the DNA methylome, of neutrophils and low density granulocytes (LDGs) in lupus patients. We studied 15 lupus patients and 15 healthy age, sex, and ethnicity matched controls. Genome-wide DNA methylation was assessed using the Illumina HumanMethylation 450 BeadChip array, which includes over 485,000 methylation sites across the entire genome. Bisulfite DNA sequencing was used to validate the array results. Statistical and bioinformatic analysis was performed to identify and characterize differentially methylated loci and genes. We identified 293 differentially methylated CG sites in neutrophils between lupus patients and controls. The majority (68%) of differentially methylated CG sites were hypomethylated in lupus neutrophils compared to controls, suggesting overall hypomethylation. We found a robust and consistent demethylation of interferon signature genes in lupus neutrophils, and similar demethylation in the same genes in autologous LDGs. Indeed, the DNA methylome in lupus neutrophils and LDGs was almost identical, suggesting similar chromatin architecture in the two granulocyte subsets. A notable exception was the hypomethylation of a CG site in the promoter region of the cytoskeleton-regulating gene RAC1 in LDGs. Our findings demonstrate a pattern of robust demethylation of interferon signature genes in lupus patients supporting a pathogenic role for neutrophils in lupus. We suggest a model whereby DNA from lupus neutrophils and LDGs externalized by NETosis enhance type-I IFN production via TLR-9 stimulation by hypomethylated DNA.

Keywords: Epigenetics; LDG; Lupus; Methylome; Neutrophils.

Figure 1

Forward and side scatter flow cytometry plots demonstrating the LDG population in a representative sample before (left) and after (right) isolation.

Figure 2

A) An overview of how the differentially methylated genes within the interferon signature group are connected in the literature to each other and to type I interferon (IFN). Thickness of the lines is proportional to how many publications mention each of the connected genes (minimum = 3 publications, maximum = 232 publications). B) The location of the 3 CG sites included on the array that were differentially methylated in MX1 between lupus and control neutrophils are depicted (1 = CG22862003, 2 = CG26312951, 3 = CG21549285). Bisulfite DNA sequencing was used to confirm and extend the results obtained from the methylation array in MX1. Bisulfite DNA sequencing primers were designed to include CG21549285, and additional 20 CG sites in its vicinity. Panel (C) shows the methylation fraction obtained from bisulfite DNA sequencing in CG21549285, and (D) shows the average methylation fraction across all 21 CG sites in lupus and control neutrophils. P values were calculated using the Mann–Whitney non-parametric test.

Figure 3

Transcriptional correlation analysis of the top 30 genes demethylated in lupus neutrophils (relative to control neutrophils). Pearson's correlation coefficients of the reported transcriptional fold changes were calculated between each of these 30 genes across 3900 public human 2-color microarray data sets. Positive correlations are shown in red and range from 1.0 (perfect positive correlation) to a minimum of −1.0. A large group of interferon-related genes can be seen here tightly clustered, indicating they are transcriptionally co-regulated.