Background/Purpose: Antiphospholipid antibodies (aPL) induce obstetric complications associated with placental insufficiency by promoting trophoblast dysfunction and inflammation at the maternal-fetal interface. Neutrophils have been found to play a critical role in aPL-mediated pregnancy loss in mice; however, the role of neutrophil extracellular traps (NETs) in obstetric APS has not been well studied. Here, we aimed to characterize the role of NETs in aPL-mediated trophoblast dysfunction and fetal loss, as well as the ability of NET inhibition to prevent these effects.

Methods: IgG was purified from patients with triple-positive APS (referred to as aPL going forward) or healthy controls. To evaluate trophoblast dysfunction, the supernatants of human neutrophils cultured with either aPL or control IgG were transferred onto human first-trimester extravillous trophoblasts (HTR-8/SVneo), and proliferation was quantified after 24 hours by BrdU. In mice (n=10-15/group), obstetric APS was modeled via intraperitoneal injection of aPL (2mg) to WT (C57BL/6) mice on days 0, 3, 6, 9, and 12 of pregnancy. On day 15, plasma was collected, and uteri were dissected to assess fetal resorption frequency. NET inhibition was modeled via knockout of the genes for neutrophil elastase (Elane-/-) or peptidylarginine deiminase 4 (Pad4-/-). An additional group of WT mice was treated with the irreversible neutrophil elastase inhibitor GW311616A. Circulating NET remnants were quantified as plasma myeloperoxidase-DNA complexes.

Results: In culture, supernatants from aPL-stimulated neutrophils significantly diminished trophoblast proliferation, compared to supernatants from control IgG-stimulated neutrophils (p=0.033) or to aPL alone (p=0.018). In the mouse model, aPL-treated mice experienced higher fetal resorption rates versus mice treated with control IgG (mean 30.1% vs. 13.1%, p=0.006). Plasma NET remnants were also higher in the aPL-treated mice (p=0.003). Compared to the 30% resorption of WT mice, Elane-/- mice (8.2% resorption, p=0.006) and Pad4-/- mice (9.6% resorption, p=0.04) were protected from aPL-mediated fetal loss. Oral treatment with the neutrophil elastase inhibitor GW311616A also prevented aPL-mediated fetal loss in WT mice (16.4% resorption; p=0.04). Importantly, there was no difference in resorption rates among these groups when treated with control IgG, suggesting a protective effect specific to aPL-mediated loss. Compared to aPL-treated WT mice, plasma myeloperoxidase-DNA complexes levels were 46% lower in aPL-treated Elane-/- mice (p=0.012) and 41.5% lower in aPL-treated Pad4-/- mice (p=0.006).

Conclusion: In addition to the direct anti-proliferative effect of aPL that has been documented by others, we found that aPL-triggered NETs further reduced normal trophoblast proliferation. In mice, we demonstrated that NET inhibition (by targeting either neutrophil elastase or PAD4) was remarkably effective in preventing aPL-mediated fetal loss and lowering circulating NET levels. Cumulatively, these data suggest that therapies targeting NETs could be effective in preventing APS obstetric morbidities and warrant further study.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-extracellular-traps-as-mediators-of-antiphospholipid-antibody-induced-trophoblast-dysfunction-and-fetal-loss/