Background/Purpose: Neutrophil hyperactivity and neutrophil extracellular trap (NET) release (NETosis) appear to play important roles in antiphospholipid syndrome (APS) pathogenesis. To kill microbes and propel NETosis, neutrophils generate reactive oxygen species (ROS) via the NADPH oxidase complex (NOX). The NADPH that fuels NOX is mostly derived from the pentose phosphate pathway (PPP). Our transcriptomic analysis of APS patient neutrophils found increased expression of PPP genes, including the instrumental gene G6PD (1.56-fold, P=5.62E^-4), which codes for glucose 6-phosphate dehydrogenase. At the same time, other groups have demonstrated that patients with severe G6PD deficiency have reduced NETosis. Here, we began to characterize how the PPP impacts neutrophils in APS.

Methods: G6PD enzyme activity was measured in patient neutrophils. Neutrophils isolated from healthy controls were stimulated with (i) IgG purified from controls (control IgG) or APS patients (APS IgG); (ii) phorbol myristate acetate (PMA, NOX-dependent NETosis); or (iii) the calcium ionophore A23187 (NOX-independent NETosis). Two PPP inhibitors were used: 6-aminonicotinamide (6-AN) and G6PDi-1, a specific inhibitor of G6PD. NETosis was quantified with SYTOX Green, and ROS production was quantified with Amplex Red. For mouse experiments, mixed-strain mice with wild-type G6PD expression were treated for 3 days with 25 mg/kg G6PDi-1 or vehicle control (n=3 mice for each treatment). After the intraperitoneal administration of APS IgG and thioglycolate, peritoneal neutrophils were isolated, and metabolic flux was assessed using a Seahorse analyzer. Finally, APS-associated thrombosis was modeled via electrolytic activation of the inferior vena cava endothelium.

Results: APS patient neutrophils (n=17, mean 21.4 mU/mL) had approximately double the G6PD enzymatic activity of healthy control neutrophils (n=11, 11.2 mU/mL, p< 0.05). In healthy control neutrophils (n=5), both 6-AN and G6PDi-1 reduced NETosis mediated by APS IgG (32% and 52%, respectively, p< 0.05) and PMA (60% and 52%, p< 0.05), but not that triggered by the calcium ionophore. Similar trends were observed for ROS production. In the studies of peritoneal mouse neutrophils, G6PDi-1 reduced APS IgG-mediated NETosis by approximately 20% (p< 0.05) and neutrophil glycolytic flux by 30% (p< 0.05). By contrast, G6PDi-1 markedly increased mitochondrial flux (340%, p< 0.05). Finally, G6PDi-1 reduced APS IgG-mediated thrombus weights by 32% (p< 0.01), which was also accompanied by a 23% reduction in circulating NET remnants (myeloperoxidase-DNA complexes, p< 0.05).

Conclusion: APS patient neutrophils have increased G6PD enzyme activity, and PPP inhibitors attenuate APS IgG-mediated human NETosis. When APS IgG was administered to mice, a G6PD inhibitor boosted neutrophil mitochondrial metabolism, while tempering neutrophil glycolytic flux, NETosis, and thrombosis. These results position the PPP as a metabolic mediator of APS-associated neutrophil hyperactivity that could be targeted clinically. Studies are now underway to further dissect the role of the PPP with G6PD-deficient mice that mimic typical human G6PD deficiency.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/modulating-pentose-phosphate-pathway-metabolism-to-temper-neutrophil-hyperactivity-in-antiphospholipid-syndrome/