Background/Purpose: Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by recurrent thrombotic events, pregnancy complications, and the presence of antiphospholipid antibodies. The pathogenesis of primary APS is incompletely understood. We aimed to investigate the role of neutrophils in primary APS.

Methods: We studied 19 patients with primary APS and 19 healthy controls matched for age, sex, and ethnicity. Neutrophils were extracted from peripheral blood samples using density gradient centrifugation followed by flow cytometry to confirm neutrophil purity. A comprehensive transcriptome analysis was performed using paired-end 100bp mRNA sequencing reads generated on an Illumina HiSeq 2000 instrument. A genome-wide DNA methylation analysis was performed using Infinium HumanMethylation450 BeadChip arrays. RNA sequencing data were normalized and analyzed following quality control assessment and filtering using EdgeR software package in the R programming environment. Differential gene expression between patients and controls was defined using a fold difference of >2 and a false discovery rate <0.01. DNA methylation analysis was performed using GenomeStudio (Illumina) and differentially methylated loci between patients and controls were identified using an absolute methylation difference of at least 10% and a P value <0.01 after correction for multiple testing. Bioinformatics analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID), and Ingenuity Pathway Analysis. 

Results: RNA sequencing analysis identified 593 overexpressed and 769 underexpressed genes in neutrophils from primary APS patients compared to age, sex, and ethnicity matched healthy controls. Neutrophils in primary APS are characterized by a pro-inflammatory phenotype defined by prominent transcriptional overexpression of interferon-regulated genes such as IFIT1 (8-fold), IFIT3 (5-fold), IFI6 (5-fold), MX1 (5-fold), HERC5 (5-fold), IFIT2 (5-fold), and ISG15 (4-fold), among many others. Neutrophils from primary APS patients are also characterized by activation of the Toll-like receptor signaling pathway with overexpression of TLR4, TLR5, and TLR8 (2-3 folds). In addition, neutrophils from patients overexpressed several Fc-gamma receptors including FCGR1A, FCGR1B, FCGR2A, FCGR3A, and FCGR3B (2.5-3.5 folds). P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin (CD62L), which play an important role in neutrophil adhesion to the vessel wall and neutrophil degranulation, were both also overexpressed in primary APS neutrophils. DNA methylation analysis in neutrophils from primary APS suggests an epigenetic architecture that is unique and very distinct from lupus neutrophils. Indeed, DNA methylation profiles in a small number of specific CG sites are very sensitive and specific to stratify patients with primary APS versus lupus with an area under the ROC curve of ≥0.90.

Conclusion: Neutrophils in patients with primary APS are activated and likely play an important role in disease pathogenesis. Further, the DNA methylation signature in primary APS neutrophils suggests a unique epigenetic architecture that is distinctly dissimilar from lupus.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophils-in-primary-antiphospholipid-syndrome-are-characterized-by-a-prominent-activated-phenotype-and-uniquely-remodeled-chromatin-architecture/