Background/Purpose:  Treatment of the thrombotic manifestations of antiphospholipid syndrome (APS) primarily focuses on inhibiting clotting pathways. In an effort to identify upstream inflammatory targets that might launch these pathways, we investigated APS patient neutrophils.

Methods:  We isolated neutrophils from the peripheral blood of 9 primary APS patients (as defined by Sydney criteria) and 9 healthy controls matched for age, sex, and ethnicity. A comprehensive transcriptome analysis was performed using paired-end 100bp mRNA sequencing reads generated on an Illumina HiSeq 2000 instrument. RNA sequencing data were normalized and analyzed using the 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. Gene ontology, pathway, and functional enrichment analyses were performed. To model APS-associated venous thrombosis in mice, IgG fractions were prepared from either healthy volunteers or patients with primary APS, who had high-titer IgG anti-β₂GPI. C57BL/6 wild-type mice were treated with control or APS IgG by intraperitoneal injection. A laparotomy was then performed, and a ligature was fastened around the inferior vena cava (IVC) to cause an 80-90% reduction in IVC blood flow. Thrombus formation in the IVC was assessed 6 hours after the procedure. Mechanistic details were further elucidated using P-selectin glycoprotein ligand 1 (PSGL-1) knockout mice, adoptive transfer of neutrophils, and an inhibitory anti-PSGL-1 antibody.

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. APS neutrophils demonstrate a pro-inflammatory phenotype with prominent transcriptional overexpression of interferon-regulated genes, the Toll-like receptor signaling pathway, Fc-gamma receptors, and adhesion molecules, including the leukocyte selectin ligand PSGL-1. The expression of neutrophil PSGL-1 was further characterized in an independent cohort of 15 primary APS patients, again revealing upregulation as compared to matched controls. In a flow-restriction model of IVC thrombosis, the introduction of APS IgG (as compared to control IgG) increases both thrombus size and thrombosis frequency in wild-type mice. While APS IgG does not accelerate thrombosis in PSGL-1 knockout mice, the phenotype can be rescued with infusion of wild-type neutrophils into PSGL-1 knockout mice. As proof of therapeutic potential, an anti-PSGL-1 monoclonal antibody inhibits APS IgG-mediated thrombosis in wild-type mice.

Conclusion:  APS neutrophils demonstrate a baseline activated phenotype, which includes the upregulation of adhesion molecules. In a model of venous thrombosis, neutrophil PSGL-1 is required for APS IgG-mediated acceleration of thrombosis. PSGL-1 represents a potential therapeutic target in APS.

« Back to 2016 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/antiphospholipid-syndrome-neutrophils-are-characterized-by-overexpression-of-p-selectin-glycoprotein-ligand-1-a-potential-therapeutic-target/