Background/Purpose: Neutrophil extracellular traps (NETs) are webs of chromatin and proteases (e.g., neutrophil elastase) that have been implicated in antiphospholipid syndrome (APS)-associated thrombosis; however, their contribution to the placental dysfunction inherent to APS remains underexplored. Here, we aimed to investigate the impact of antiphospholipid antibodies (aPL) and downstream NET formation on implantation, placental health, and fetal viability in mice.

Methods: Wild-type (WT) mice were injected with 2 mg of IgG pooled from either healthy donors or APS patients on gestational days 0, 3, 6, 9, and 12; placental resorptions were assessed on day 15. Pad4-deficient mice, which are known to have reduced NET formation, were evaluated in parallel. Circulating myeloperoxidase (MPO)-DNA complexes were quantified as markers of NETs, and plasma PIGF-2 as a measure of placenta-associated angiogenesis. Placental histopathology was assessed using a semiquantitative scoring system evaluating six parameters—vasculopathy, thrombosis, inflammation, fibrin deposition, villous ischemia, and hemorrhage—each graded from 0 (normal) to 3 (severe). Placental expression of citrullinated-histone H3 was analyzed by immunoblotting. In vitro experiments were performed with HTR-8/SVneo trophoblasts.

Results: Among APS IgG-treated WT mice, 29.6% with a vaginal plug (indicating copulation) had a false pregnancy, compared with 16.0% in the control IgG group. None of the Pad4-/- mice treated with APS IgG had a false pregnancy. Mean fetal resorption rates were significantly higher in APS IgG-treated WT mice (15.8% for n=16 mice) compared with control IgG-treated WT mice (5.8% for n=19 mice, p=0.0096). APS IgG-treated Pad4-/- mice had an average of 3.6% resorptions (n=10 mice, p=0.0079 compared with APS WT mice). Plasma MPO-DNA complexes were significantly higher in APS WT mice compared with both controls (mean 1.8-fold, p=0.0061) and APS Pad4-/- mice (1.9-fold, p=0.0135). In contrast, plasma PIGF-1 levels were reduced by approximately 2-fold in APS WT mice compared with controls (p=0.0100); the APS Pad4-/- mice did not differ from controls. Inflammation was the most prominent feature in non-resorbed APS WT placentas (mean score=2.7 versus 1.0 in controls), followed by vasculopathy and hemorrhage; the inflammatory infiltrate was predominantly neutrophilic. Notably, resorbed APS WT placentas had dramatically increased citrullinated-histone H3 expression (a marker of NETs), which was absent in resorbed control placentas. Finally, in vitro, trophoblast proliferation was impaired after 48 hours of exposure to purified NETs (1 µg/mL) compared with vehicle-treated cells (p = 0.0323). The neutrophil elastase inhibitor GW311616A restored proliferation to baseline levels, suggesting a role for elastase’s enzyme activity in NET-mediated trophoblast dysfunction.

Conclusion: These findings suggest that APS IgG may contribute to fetal loss and placental alterations by triggering increased NET formation. Significant protection in NET-deficient Pad4-/- mice suggests that NET inhibition is an approach worth exploring to improve pregnancy outcomes in the APS clinic.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/trophoblast-dysfunction-and-placental-alterations-in-a-mouse-model-of-antiphospholipid-syndrome-the-potential-role-of-neutrophil-extracellular-traps/