Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target
Jason S. Knight, He Meng, Patrick Coit, Srilakshmi Yalavarthi, Gautam Sule, Alex A. Gandhi, Robert C. Grenn, Levi F. Mazza, Ramadan A. Ali, Paul Renauer, Jonathan D. Wren, Paula L. Bockenstedt, Hui Wang, Daniel T. Eitzman, Amr H. Sawalha
Jason S. Knight, He Meng, Patrick Coit, Srilakshmi Yalavarthi, Gautam Sule, Alex A. Gandhi, Robert C. Grenn, Levi F. Mazza, Ramadan A. Ali, Paul Renauer, Jonathan D. Wren, Paula L. Bockenstedt, Hui Wang, Daniel T. Eitzman, Amr H. Sawalha
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Research Article

Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target

Abstract

Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils — neutrophil extracellular traps (NETs), in particular — in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1–KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1–deficient mice by infusion of WT neutrophils, while an anti–PSGL-1 monoclonal antibody inhibited APS IgG–mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS.

Authors

Jason S. Knight, He Meng, Patrick Coit, Srilakshmi Yalavarthi, Gautam Sule, Alex A. Gandhi, Robert C. Grenn, Levi F. Mazza, Ramadan A. Ali, Paul Renauer, Jonathan D. Wren, Paula L. Bockenstedt, Hui Wang, Daniel T. Eitzman, Amr H. Sawalha

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Figure 3

PSGL-1 is upregulated in APS patient neutrophils.

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PSGL-1 is upregulated in APS patient neutrophils. (A) RNA was prepared f...
(A) RNA was prepared from patients with primary APS, none of whom were a part of the RNA-sequencing analysis. Expression of the gene encoding for PSGL-1 was determined by quantitative PCR, with normalization to the β-actin gene. The median for each group is denoted by a solid horizontal line, while each data point represents a unique control/patient; **P < 0.01 by Mann-Whitney test. (B) Neutrophils were isolated from healthy controls and then incubated with heterologous control serum or APS serum for 4 hours. RNA was prepared and subjected to quantitative PCR as in panel A. The median for each group is denoted by a solid horizontal line, while each data point represents stimulation with a unique control/patient serum sample; *P < 0.05 by Mann-Whitney test. (C) Control neutrophils were incubated with either APS serum (n = 5) or the same APS serum samples depleted of total IgG. After 4 hours, RNA was prepared and subjected to quantitative PCR. Box-and-whisker plots denote minimum, 25th percentile, median, 75th percentile, and maximum; **P < 0.01 by paired t test. (D) Control neutrophils were incubated with IgG purified from controls or APS patients (n = 5). After 4 hours, RNA was prepared and subjected to quantitative PCR. *P < 0.05 by two-tailed t test.