Session Type: Abstract Session
Session Time: 9:00AM-9:15AM
Background/Purpose: Neutrophil hyperactivity and neutrophil extracellular trap (NET) release (NETosis) appear to play roles in APS pathophysiology. Until recently, it was thought that neutrophils relied primarily on glucose uptake and glycolysis to fulfill their energy needs. Accumulating evidence now suggests that metabolic plasticity guides both physiologic and pathologic neutrophil functions. Our previous work characterizing the transcriptome of APS neutrophils found that genes related to glycogenolysis and the pentose phosphate pathway were markedly upregulated. We now begin to characterize how neutrophil metabolism may impact APS-relevant NETosis.
Methods: Primary human neutrophils were stimulated with phorbol myristate acetate (PMA), calcium ionophore A23187 (Ca iono), or affinity-purified anti-β2GPI IgG (aβ2GPI). PMA and Ca iono were chosen as mechanistic anchors given their divergent mechanisms for triggering NETosis (NADPH oxidase-dependent and -independent, respectively). Inhibitors included 2-deoxy-D-glucose (2-DG, a competitive inhibitor of glycolysis) and 6-aminonicotinamide (6-AN, a glucose-6-phosphate dehydrogenase inhibitor that blocks the pentose phosphate pathway). Metabolic flux analysis was conducted using a Seahorse analyzer. NETosis was quantified with SYTOX Green. APS-associated thrombosis was modeled in C57BL/6 mice via electrolytic activation of the inferior vena cava (IVC) endothelium.
Results: In the metabolic flux analysis, Ca iono triggered an earlier peak in oxygen consumption than did PMA (30 min vs. 90 min). Peak Ca iono-induced oxygen consumption decreased by 30% with 2-DG and by 74% with 6-AN. For PMA, the decreases were 39% (2-DG) and 47% (6-AN). Interestingly, as compared with healthy control neutrophils, neutrophils isolated from primary APS patients (n=3) demonstrated a pattern of oxygen consumption indicative of more robust mitochondrial reserve. When NETosis was considered, PMA-induced NETosis was sensitive to both 2-DG (46% reduction) and 6-AN (57% reduction). In contrast, Ca iono-induced NETosis was insensitive to both inhibitors. Importantly, aβ2GPI-induced NETosis mirrored PMA and was sensitive to both 2-DG (< 0.05) and 6-AN (p< 0.05). In a mouse model of APS, transfer of IgG from APS patients into mice (n=8-10/group) doubled IVC thrombus size (mean 9.7 mm vs. 5.1 mm) at 24 hours. When APS mice were administered daily 2-DG (250-1000 mg/kg) for two weeks prior to thrombus induction, thrombus size was decreased back to the baseline of 5 mm (p< 0.0001); this was accompanied by a reduction in plasma NET remnants.
Conclusion: We demonstrate for the first time that inhibitors of glycolysis and the pentose phosphate pathway attenuate aβ2GPI-induced NETosis. Furthermore, neutrophils isolated from patients with primary APS appear to have baseline characteristics suggestive of a more robust mitochondrial network, which may hint at metabolic plasticity in vivo. The potential clinical applicability of these studies are emphasized by the ability of 2-DG to restrain APS-associated thrombosis in mice. Numerous studies are now underway to further dissect these metabolic (and potentially modifiable) underpinnings of APS pathophysiology.
To cite this abstract in AMA style:Tambralli A, Harbaugh A, Estes S, Yalavarthi S, Hoy C, Sule G, Knight J. Immunometabolism of Neutrophils in Antiphospholipid Syndrome (APS) [abstract]. Arthritis Rheumatol. 2021; 73 (suppl 10). https://acrabstracts.org/abstract/immunometabolism-of-neutrophils-in-antiphospholipid-syndrome-aps/. Accessed July 4, 2022.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immunometabolism-of-neutrophils-in-antiphospholipid-syndrome-aps/