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.

IFI44L promoter methylation as a blood biomarker for systemic lupus erythematosus

“…All of the differentially methylated CpGs in IFI44L were located within 1500 base-pairs upstream of the transcription start site and the 5′-untranslated region, including cg00855901, cg03607951, cg06872964, cg17980508, cg13304609 and cg05696877 (see online supplementary table S4), among which cg06872964 is one of very significant differentially methylated CpG sites comparing patients with SLE with HCs (Δβ SLEa vs HC =−0.31 and Δβ SLEi vs HC =−0.30) (see online supplementary figure S1). In addition, our previous studies have revealed significant hypomethylation of the IFI44L promoter in naive CD4 + T cells and neutrophils of patients with SLE compared with HCs 19 21. Therefore, we assessed the diagnostic value of IFI44L promoter methylation level in SLE using peripheral blood samples.…”

Neutrophil heterogeneity: implications for homeostasis and pathogenesis

“…23 Thus, this evidence supports an inducible epigenetic and genetic regulation of neutrophil gene expression. Moreover, the association between genetic variants 24 and altered methylation profiles 25 in neutrophils associated with disease susceptibility emphasizes the importance of understanding how gene expression is controlled in neutrophils.…”

Aberrant Epigenetic Regulation in the Pathogenesis of Systemic Lupus Erythematosus and Its Implication in Precision Medicine

“…The authors found that the highest DNA methylation variation lay in gene body and upstream regions, whereas the lowest was observed in promoters. The iVMFs were mainly associated with tran- Kaplan et al, 2004Javierre and Richardson, 2011Lu et al, 2007Oelke et al, 2004Lu et al, 2002Coit et al, 2013Zhao et al, 2010bHedrich et al, 2014 B cells CD5 HRES1 hypomethylation increased B cell activation induction of cross-reactive autoantibodies Garaud et al, 2009Fali et al, 2014 PBMCs IFNGR2 LCN2 hypomethylation lymphadenopathy and kidney damage a common marker for lupus nephritis Schwarting et al, 1998;Javierre et al, 2010Javierre et al, 2010 Whole blood IFI44L a highly specific and sensitive biomarker Zhao et al, 2016b Neutrophils MX1, IFI44L, IFITM1, PARP9, IFIT3, DDX60, LY6E, ISG15 hypomethylation increased production of type I IFN driving abnormal B cell differentiation Coit et al, 2015b Zhan/Guo/Lu scription regulation, responsive function, and signal transduction pathways, and positively correlated with inclusion of the exon when found at differentially expressed exons. Neutrophils are also found to play an important role in the pathogenesis of lupus.…”

“…Neutrophils are also found to play an important role in the pathogenesis of lupus. Coit et al [2015b] recently performed an epigenome-wide study between lupus patients and controls and identified 293 differentially methylated CG sites in neutrophils, of which the majority (68%) were hypomethylated. Among the differentially methylated sites, IFN signature genes were found demethylated in a robust and consistent way in lupus neutrophils compared to controls ( table 1 ), suggesting a pathogenic role for neutrophils in lupus.…”