Background/Purpose: As early effectors of the immune response, neutrophils must access energy rapidly. Neutrophils rely on glycolysis for antimicrobial functions such as phagocytosis and reactive oxygen species (ROS) production. The glycolytic metabolite, glucose-6-phosphate, is a substrate for the pentose phosphate pathway (PPP), which is also critical for ROS production by neutrophils. We have previously found that antiphospholipid syndrome (APS)-associated neutrophil extracellular trap (NET) formation depends on glycolysis and the PPP, with these pathways especially active in APS patients with microvascular manifestations (i.e., history of diffuse alveolar hemorrhage, thrombotic microangiopathy, or catastrophic APS). Here, we conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics to further delineate the metabolomic landscape of microvascular APS neutrophils.

Methods: Circulating markers of NETs and neutrophil activation were measured in the plasma of microvascular APS patients and matched controls by ELISA. Neutrophil glycolytic flux was determined using a Seahorse extracellular flux analyzer. Polar metabolites were collected from isolated neutrophils with 80% methanol. An Agilent 1290 UHPLC and a 6490 Triple Quadrupole (QqQ) mass spectrometer were used for label-free metabolomics analysis to assay >200 metabolites in central carbon metabolism. A noise cut-off value of 3,000 (peak area) was chosen by manual inspection of the distributions of the coefficients of variation. Each sample was median normalized based on the total intensity of all metabolites. A two-tailed t-test with p< 0.1 was used to identify significant metabolites.

Results: As compared with control plasma (n=16), microvascular APS plasma (n=10) had more myeloperoxidase-DNA complexes (a marker of NETs; mean 0.23 vs 0.40 mg/mL, p< 0.01) and calprotectin (a marker of neutrophil activation and NETs, mean 204 vs 695 ng/mL, p< 0.01). As compared with control neutrophils, microvascular APS neutrophils had a 1.6-fold greater glycolytic capacity (p< 0.05). When we assessed neutrophil glycolytic and mitochondrial metabolites, we found that glucose-6-phosphate (p< 0.05) and alpha-ketoglutaric acid (aKG, p< 0.1) were higher in microvascular APS neutrophils than controls. In contrast, succinic acid (p< 0.05) and malic acid (p< 0.01) were lower in microvascular APS neutrophils than controls.

Conclusion: As expected, APS patients with microvascular manifestations have more circulating NETs and neutrophil glycolytic flux than controls. Their neutrophil metabolome is characterized by increases in the key glycolytic metabolite glucose-6-phosphate and the upper citric acid cycle metabolite aKG. In contrast, succinic acid and malic acid, two metabolites in the lower citric acid cycle, are reduced in microvascular APS. This suggests that the citric acid cycle, with an inflection point at aKG, is dysfunctional in microvascular APS neutrophils, potentially shifting the cells toward increased reliance on pro-inflammatory glycolysis. Experiments are underway to determine the effects of citric acid cycle metabolites on APS-associated glycolytic flux and NETosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/metabolomic-profiling-reveals-a-role-for-citric-acid-cycle-dysfunction-in-antiphospholipid-syndrome-neutrophils/