Adequate iron availability during pregnancy is essential for fetal development and maternal health. Iron deficiency and its most common manifestation, anemia, are highly prevalent during pregnancy and can have adverse effects on the mother and the fetus. Systemic maternal inflammation during pregnancy is also quite common, and either results from infections (bacterial, viral, parasitic), or accompanies chronic conditions including obesity, diabetes, and autoimmune diseases. Adverse outcomes associated with inflamed pregnancies include spontaneous abortion, preterm labor, intrauterine growth restriction, fetal death, gross developmental abnormalities and autistic behaviors in offspring. Our preliminary studies in mice demonstrated a surprising synergistic adverse interaction between maternal iron deficiency and inflammation that led to embryotoxicity which was not observed with either condition alone. Compared to dams with normal iron status, those that were iron-deficient had significantly higher frequency of embryo abnormalities in the setting of acute maternal inflammation caused by LPS injection. We aim to define the underlying mechanisms of this synergy: Aim 1. Determine the effect of iron deficiency on maternal, placental and embryo inflammation. Using our mouse models of LPS-triggered inflammation, we will examine whether iron deficiency in pregnancy enhances pro-inflammatory response or alters immune cell census in the mother, placenta and embryo. Aim 2. Characterize the iron-dependent regulation of TNF receptor 1 Based on the strong correlation between placental TNFR1 and TFR1 in both humans and mice, we hypothesize that cellular iron status regulates TNFR1 levels. We will define the specific placental cell type that increases TNFR1 in response to iron deficiency, and examine candidate mechanisms regulating TNFR1. . Aim 3. Determine the role of the TNF pathway in adverse synergy in vivo—Our preliminary data implicated TNFα-TNFR pathway in mediating adverse outcomes in iron-deficient inflamed pregnancies. We will use mice with endothelial- or trophoblast-specific deficiency of TNFR1 to determine whether the TNF pathway is required for the adverse synergy in the model of acute inflammation. Aim 4. Define the effect of iron deficiency on placental cellular transcriptome. We will complement our candidate-driven approaches with an unbiased single-cell RNAseq on mouse placentas from iron-deficient and iron-replete pregnancies treated with LPS to gain detailed insight into how iron deficiency alters placental cell populations as well as their transcriptional activity in response to inflammation. Our proposal analyzes a novel interaction between iron deficiency and inflammation. Considering the high global prevalence of this combination of disorders in pregnant women, the subject has outstanding translational importance, and our studies could help explain commonly observed adverse outcomes. Long-term, our findings have the potential to influence ...