Iron-Dependent Apoptosis Causes Embryotoxicity in Inflamed and Obese Pregnancy

Abstract

Since iron is essential for pregnancy, iron supplements are routinely prescribed to pregnant women without screening for iron deficiency. Most women in developed countries have sufficient iron stores, prompting concerns of the potential risks of iron supplementation in iron-replete women. A signal of potential harm of this practice is the association of maternal iron marker ferritin and pregnancy complications. Since ferritin is also induced by inflammation, it is unclear whether iron excess, inflammation, or their interaction cause fetal injury. We used mouse models to address this question. Two mouse models of maternal iron excess were used and compared to mice with normal iron status. To model dietary iron supplementation, C57BL/6 females were fed high-iron diet (2500-5000ppm iron) for 1 week prior to and during pregnancy. To model genetic iron loading, hepcidin knockout females were used and fed standard chow (185ppm iron). Two models of maternal inflammation were used. Acute systemic maternal inflammation was induced by a single subcutaneous injection of LPS on E8.5 or 15.5. Obesity was used as a model of mild chronic systemic maternal inflammation and was induced by feeding female mice Western diet. We evaluated the maternal, placental, and embryo response to iron excess in LPS- and obesity-induced inflammation at various time points. In the model of acute inflammation, we observed an adverse synergistic effect on embryo development when dams were both iron-loaded and LPS-injected, but not with either condition alone. On E8.5, LPS injection in iron-loaded dams caused embryo loss and encephalic malformations (Fig 1a) and on E15.5, LPS injection in iron-loaded dams caused embryo demise and resorption (Fig 1b). Western blotting and immunostaining showed increased apoptotic marker cleaved caspase-3 protein localized to endothelia in placentas and embryos, only when the dam was both iron-loaded and LPS-injected. Cytokine screen in vitro identified TNFα as the signal that synergized with iron to potentiate apoptosis of endothelial cells. Treatment of iron-loaded dams with TNFα-neutralizing antibody protected embryos from LPS-induced death, demonstrating the embryotoxic role of maternal TNFα in iron-loaded dams. Furthermore, RNA-Seq analysis of placental endothelial cells showed enrichment in oxidative stress pathways with maternal iron loading. Treatment of iron-loaded dams with antioxidant α-tocopherol protected against LPS-induced embryo death, attenuated placental inflammation and cleaved caspase-3 expression, and prevented endothelial apoptosis in the placenta and embryo. These data show that iron loading causes endothelial oxidative stress, which sensitizes endothelial cells to inflammation-induced apoptosis. In the obesity model, iron loading worsened embryotoxicity, causing subcutaneous hemorrhaging and eye malformations (Fig 1c), and potentiated placental cleaved caspase-3 expression. Iron loading increased TNFα levels in maternal serum. Importantly, treatment of obese iron-loaded dams with neutralizing TNFα antibody throughout pregnancy reduced the incidence of embryo eye malformations, confirming the role of TNFα in causing embryo injury in iron-loaded obese pregnancy. In summary, maternal iron excess worsened inflammation-induced embryo injury. Iron-dependent embryotoxicity was mediated by TNFα causing lethal apoptotic damage to embryo and placental endothelium which was prevented by antioxidant therapy. Our models raise the possibility that women exposed to both excessive iron and inflammation could be at risk for preventable pregnancy complications. Figure 1. Embryo outcomes in mouse pregnancies with maternal iron loading and inflammation. Figure 1 Disclosures Ganz: Akebia: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company; Global Blood Therapeutics: Consultancy; Ambys: Consultancy; Gossamer Bio: Consultancy; Astellas: Consultancy; Rockwell: Consultancy; Sierra Oncology: Consultancy; Disc Medicine: Consultancy; American Regent: Consultancy. Nemeth:Intrinsic LifeSciences: Current equity holder in private company; Vifor: Consultancy; Protagonist: Consultancy; Silarus Therapeutics: Current equity holder in private company; Ionis Pharmaceuticals: Consultancy.