Background/Purpose: Malleable metabolic pathways guide both productive and pathological neutrophil functions, including neutrophil extracellular trap release (NETosis). NETosis plays an important role in APS pathophysiology. Explicit glycolysis inhibitors such as 2-deoxy-D-glucose (2-DG) negate many forms of NETosis (including that mediated by anti-beta-2 glycoprotein I antibodies). During fasting and in response to certain dietary modifications, fatty acids can be converted into soluble ketones that can be used as a source of energy. While ketone bodies modulate both glycolytic and mitochondrial metabolism, their impact on NETosis has only been minimally studied.

Methods: In glucose-sufficient media, human neutrophils were treated with the following stimuli (n=4-5 per condition): phorbol myristate acetate (PMA), calcium ionophore A23187 (Ca iono), and IgG fractions from either triple-positive APS patients (APS IgG) or controls (control IgG). Tested inhibitors included 2-DG (glycolysis), 6-aminonicotinamide (6-AN, pentose phosphate pathway), and beta-hydroxybutyrate (a physiological ketone body). NETosis was quantified with SYTOX Green. Reactive oxygen species (ROS) production was quantified with Amplex Red. Cellular bioenergetics were assessed using the Seahorse Extracellular Flux Analyzer.

Results: From the perspective of metabolism, NETosis can be triggered by two distinct pathways represented by PMA (glycolysis- and NADPH oxidase-dependent NETosis) and Ca iono (glycolysis- and NADPH oxidase-independent). Here, NETosis induced by PMA (4.4-fold), Ca iono (3.7-fold), and APS IgG (2.7-fold) was higher than that induced by control IgG (all p< 0.05). Both 2-DG and 6-AN reduced NETosis triggered by PMA (39% and 59%, respectively; p< 0.05) and APS IgG (41% and 35%; p< 0.05), but not by Ca iono. In contrast, when beta-hydroxybutyrate was tested as an inhibitor, NETosis was potently diminished for all three of PMA (79%, p< 0.05), APS IgG (63%, p< 0.05), and Ca iono (65%, p< 0.05). While stimulation with control IgG and Ca iono led to only low levels of cellular ROS, both PMA (3.4-fold, p< 0.05) and APS IgG (2.8-fold, p< 0.05) led to potent ROS production. Not only 2-DG and 6-AN, but also beta-hydroxybutyrate (70%, p< 0.05), robustly suppressed APS IgG-induced neutrophil ROS. When quantified with the Seahorse analyzer, neutrophil maximal extracellular acidification rate (a surrogate for glycolysis) and maximal oxygen consumption rate were massively reduced ( >90% and >70%, respectively) for all stimuli in the presence of beta-hydroxybutyrate.

Conclusion: We found, for the first time to our knowledge, that the physiological ketone body beta-hydroxybutyrate inhibits both NADPH oxidase-dependent and -independent NETosis, potentially targeting multiple NETosis pathways and thereby limiting escape mechanisms. Furthermore, APS IgG-induced NETosis was highly susceptible to the effects of beta-hydroxybutyrate, laying the foundation for examining ketosis as a possible treatment paradigm. Experiments, including mouse thrombosis models, are now underway to better understand the therapeutic potential suggested by these preclinical data.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-physiological-ketone-body-beta-hydroxybutyrate-potently-neutralizes-aps-associated-netosis/