Background/Purpose: Neutrophil hyperactivity and neutrophil extracellular trap (NET) release (NETosis) contribute to antiphospholipid syndrome (APS) pathogenesis. We recently discovered that APS patient neutrophils have more glycolytic flux than healthy neutrophils and that inhibiting glycolysis in mice reduces neutrophil hyperactivity and thrombosis. In addition, our transcriptomic analysis of APS neutrophils identified regulation of autophagosome assembly (p=1.9E-5) and ubiquitin-protein ligase activity (p=0.0057) as among the top-upregulated pathways. It has previously been shown that dysfunctional mitochondrial autophagy (mitophagy) can enhance glycolysis across various disease contexts. Here, we began to characterize the potential influence of dysfunctional neutrophil mitophagy on the pathogenesis of APS by determining its role in augmenting glycolysis, NETosis, and thrombosis.

Methods: For patient profiling, neutrophils were freshly isolated from healthy controls (n=8) or individuals with primary APS (n=11). For in vitro studies, control neutrophils were stimulated with IgG pooled from either controls or individuals with APS who had high-titer anti-β2GPI IgG. Wild-type C57BL/6 (WT) mice and mitophagy-deficient Pink1-/- mice were used for animal studies. Thioglycolate-elicited mouse neutrophils were isolated from the peritoneum 16 hours after administration of IgG. Neutrophil mitophagy (MtPhagy), mitochondrial polarization (JC-10), and mitochondrial reactive oxygen species (MitoSOX) were assessed by flow cytometry. NETosis was quantified with SYTOX Green, and glycolytic capacity (the maximal cellular utilization of glycolysis) was assessed using a Seahorse metabolic flux analyzer. Thrombosis was modeled via electrolytic activation of the mouse inferior vena cava.

Results: Compared with controls, APS patients had fewer neutrophils undergoing beneficial mitophagy (66% vs 28%, p< 0.05). In addition, APS patients had more neutrophils with dysfunctional, depolarized mitochondria (11% vs 20%, p< 0.01) and produced more harmful mitochondrial reactive oxygen species (1.3-fold, p< 0.05). The mitophagy activator valinomycin reduced human APS IgG-mediated NETosis to levels seen with control IgG (p< 0.05). Further, compared with neutrophils from WT mice, neutrophils from mitophagy-deficient Pink1-/- mice (n=3 mice for each genotype) had increased glycolytic capacity (1.3-fold, p< 0.05) and augmented APS IgG-mediated NETosis (1.4-fold, p< 0.01). Finally, when APS-associated thrombosis was modeled in mice (n=9 WT and n=5 Pink1-/-), Pink1-/- mice developed significantly larger thrombi (mean 5.1 mg vs 8.0 mg, p< 0.01).

Conclusion: APS patient neutrophils demonstrated defective mitophagy and dysfunctional mitochondria. Mice with a baseline defect in mitophagy (Pink1-/-) phenocopied APS patients by demonstrating more glycolytic flux, NETosis, and thrombosis. Notably, activating mitophagy tempered APS IgG-mediated human NETosis. These results identify dysfunctional mitophagy as a driver of neutrophil hyperactivity and thrombosis in APS, and restoring normal mitophagy could be a promising approach for a future therapeutic intervention.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dysfunctional-mitophagy-propels-neutrophil-hyperactivity-and-thrombosis-in-antiphospholipid-syndrome/