Elsevier

Journal of Autoimmunity

Volume 58, April 2015, Pages 59-66
Journal of Autoimmunity

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

https://doi.org/10.1016/j.jaut.2015.01.004Get rights and content

Highlights

  • This is the first epigenetic study in lupus neutrophils and low density granulocytes (LDGs).

  • Interferon-regulated genes are strongly demethylated in lupus neutrophils.

  • The DNA methylome of LDGs is almost identical to mature neutrophils in lupus.

  • The cytoskeleton-related gene RAC1 is hypomethylated in lupus LDGs compared to 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.

Introduction

Systemic lupus erythematosus is a chronic relapsing autoimmune disease characterized by autoantibody production and multi-organ involvement. The etiology of lupus is incompletely understood. However, a number of environmental triggers and epigenetic factors, in the setting of a genetic susceptibility background, are thought to contribute to the etiology of the disease [1]. Gene expression profiling in peripheral blood mononuclear cells (PBMCs) from patients with lupus demonstrates a robust and confirmed interferon signature [2]. In addition, a granulocyte expression signature has been observed [3], suggesting a role for neutrophils in the pathogenesis of lupus. Recent evidence indicates that PBMCs from lupus patients include a subset of “immature” or “abnormal” proinflammatory neutrophils with a lower density, which have been called low density granulocytes (LDGs) [4]. This neutrophil subset purifies with PBMCs upon density gradient centrifugation and explains the granulocyte signature observed in lupus PBMCs [3]. Subsequently, it was noted that this granulocyte subset is more prone to a recently described neutrophil cell death process by forming neutrophil extracellular traps (NETs) [5]. Indeed, NETs formation stimulates interferon alpha production from plasmacytoid dendritic cells, thereby contributing to the pathogenesis of lupus [6]. Evidence suggests that lupus neutrophils, and LDGs in particular, play an important role in tissue damage (including glomerulonephritis and lupus skin involvement), as well as endothelial damage in lupus patients [5]. Several studies have also suggested altered functional capacity of lupus neutrophils compared to healthy controls [6].

Despite the recent and important progress in recognizing the potential role of neutrophils in the pathogenesis of lupus, there remain significant knowledge gaps that require further investigation. DNA methylation changes play an important role in the pathogenesis of lupus. To understand the epigenetic accessibility that underlies the pathogenic role of neutrophils in lupus, we performed a genome-wide DNA methylation study in normal density lupus neutrophils (hereafter referred to as neutrophils) compared to age, sex, and ethnicity matched healthy controls, and in normal density lupus neutrophils compared to autologous LDGs.

Section snippets

Lupus patients and controls

We included 15 female lupus patients and 15 age (±5 years), sex, and ethnicity matched healthy controls in this study (Table 1). The average age for patients and controls was 36.9yr ± 10.2 and 38.7yr ± 9.6, respectively (P = 0.62). All patients studied fulfilled the American College of Rheumatology (ACR) classification criteria for lupus, and were recruited from the University of Michigan rheumatology clinics or the Lupus Natural History Protocol at the National Institute of Arthritis and

Results

We observed significant and consistent DNA methylation differences between neutrophils in lupus patients when compared to age, sex, and ethnicity matched healthy controls. There were a total of 293 differentially methylated CG sites, with 199 (68%) hypomethylated and 94 (32%) hypermethylated in lupus neutrophils (Supplementary Table S1). The top hypomethylated and hypermethylated sites are listed in Table 2. Raw and normalized DNA methylation data were deposited in Gene Expression Omnibus (GEO

Discussion

The role of components of the innate immune response in the pathogenesis of systemic autoimmune diseases, such as lupus, has been increasingly recognized [15], [16]. Lupus is characterized by activation of plasmacytoid dendritic cells, a robust type-I interferon expression signature, and hyper-responsiveness of PBMCs to type-I interferon [17], [18]. Indeed, T cells in lupus patients are characterized by a permissive epigenetic architecture in interferon-regulated genes, which precedes active

Conclusion

We characterized the DNA methylome of lupus neutrophils for the first time. We showed evidence for robust but identical level of DNA demethylation in interferon-regulated genes in lupus neutrophils and LDGs. Our data suggest that neutrophils and LDGs in lupus patients are highly identical at the epigenetic level, with the notable exception of the cytoskeleton regulating gene RAC1, which is demethylated in at least 1 CG site in lupus LDGs compared to autologous neutrophils. Importantly, we

Competing interests

None declared.

Acknowledgments and funding

Research reported in this publication was supported by the Lupus Research Institute, the Intramural Research Program, NIAMS, NIH, and the National Institute of Allergy and Infectious Diseases, NIH under award number R01AI097134.

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