Preeclampsia (PE) is a disorder of pregnancy that manifests as late gestational maternal hypertension and proteinuria and can be life-threatening to both the mother and baby. It is believed that abnormal placentation is responsible for the cascade of events leading to the maternal syndrome. Embryo implantation is critical to establishing a healthy pregnancy. Defective implantation can cause adverse “ripple effects,” leading to abnormal decidualization and placentation, retarded fetal development, and poor pregnancy outcomes, such as PE and fetal growth restriction. The precise mechanism(s) of implantation defects that lead to PE remain elusive. BPH/5 mice, which spontaneously develop the cardinal features of PE, show peri-implantation defects including upregulation of Cox2 and IL-15 at the maternal-fetal interface. This was associated with decreased decidual natural killer (dNK) cells, which have important roles in establishing placental perfusion. Interestingly, a single administration of a Cox2 inhibitor (celecoxib) during decidualization restrained Cox2 and IL-15 expression, restored dNK cell numbers, improved fetal growth, and attenuated late gestational hypertension in BPH/5 female mice. This study provides evidence that decidual overexpression of Cox2 and IL-15 may trigger the adverse pregnancy outcomes reflected in the preeclamptic syndrome, underscoring the idea that Cox2 inhibitor treatment is an effective strategy for the prevention of PE-associated fetal and maternal morbidity and mortality.

Macrophages participate in immunity, tissue repair and tissue homeostasis. Activation of Toll-like receptors (TLRs) by conserved exogenous or endogenous structures initiates signaling cascades that result in the release of cytokines such as tumor necrosis factor α (TNFα). Extracellular substrate stiffness is known to regulate functions of non-immune cells through a process called mechanotransduction, yet less is known about how physical cues affect macrophage function or TLR signaling. To investigate this question, we cultured murine primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells on fibronectin-coated polyacrylamide (PA) gels of defined stiffnesses (1, 20 and 150 kPa) that approximate the physical properties of physiologic tissues. BMMs on all gels were smaller and more circular than those on rigid glass. Macrophages on intermediate stiffness 20 kPa PA gels were slightly larger and less circular than those on either 1 or 150 kPa. Secretion of the pro-inflammatory cytokine, TNFα, in response to stimulation of TLR4 and TLR9 was increased in macrophages grown on soft gels versus more rigid gels, particularly for BMMs. Inhibition of the rho-associated coiled-coil kinase 1/2 (ROCK1/2), key mediators in cell contractility and mechanotransduction, enhanced release of TNFα in response to stimulation of TLR4. ROCK1/2 inhibition enhanced phosphorylation of the TLR downstream signaling molecules, p38, ERK1/2 and NFκB. Our data indicate that physical cues from the extracellular environment regulate macrophage morphology and TLR signaling. These findings have important implications in the regulation of macrophage function in diseased tissues and offer a novel pharmacological target for the manipulation of macrophage function in vivo.

Preeclampsia is a devastating complication of pregnancy characterized by late-gestation hypertension and proteinuria. Because the only definitive treatment is delivery of the fetus and placenta, preeclampsia contributes to increased morbidity and mortality of both mother and fetus. The BPH/5 mouse model, which spontaneously develops a syndrome strikingly similar to preeclampsia, displays excessive inflammation and suppression of inflammation improves pregnancy outcomes. During early pregnancy, decidual macrophages play an important role in promoting maternal tolerance to fetal antigens and regulating tissue remodeling, two functions that are critical for normal placental development. BPH/5 pregnancies are characterized by abnormal placentation; therefore, we hypothesized that macrophage localization and/or function is altered during early pregnancy at the site of placental formation (the decidua) compared to C57BL/6 controls. At early gestation time points, before the onset of maternal hypertension or proteinuria, there was a reduction in the number of macrophages in BPH/5 decidua and a concomitant increase in activated T cells compared to C57BL/6. BPH/5 decidua also exhibited decreased expression of the immunosuppressive cytokine, IL-10, and increased expression of pro-inflammatory, inducible nitric oxide synthase (iNOS). Together, these data suggest that a reduction in decidual macrophages during pregnancy is associated with immune activation in BPH/5 mice, inadequate placental development and may contribute to adverse pregnancy outcomes in this model.

Preeclampsia (PE) is a hypertensive disease that affects up to 10% of pregnancies worldwide. Although a leading cause of maternal and fetal morbidity/mortality, the cause is unknown. Inadequate remodeling of decidual vessels and reduced placental perfusion are hallmarks of PE. Decidual Natural Killer cells (dNKs) are critical in this process. Early in pregnancy, dNKs are activated by uterine IL-15 to promote angiogenesis at the maternal-fetal interface. Activated dNKs produce IFN-γ to facilitate vasodilation of decidual vessels. In the BPH/5 mouse model that spontaneously develops late gestation hypertension, proteinuria and placentopathies, we have demonstrated an early pregnancy reduction in dNK activation and IFN-γ mRNA at the maternal-fetal interface. We functionally linked uterine IL-15 overexpression to dNK loss in C57 mice. Here we tested the hypothesis that uterine IL-15 in BPH/5 mice contributes to dNK dysregulation at the maternal-fetal interface prior to placenta formation. BPH/5 e7.5 implantation sites show a 5-fold increase in IL-15 protein vs C57 (.7±.07vs.35±.17, n=4, p<.05). We have shown that Cox2 inhibition early in BPH/5 pregnancy improves fetoplacental development. Cox2-derived products influence expression of the pro-inflammatory cytokine, IL-15. To address if Cox2 inhibition alters IL-15 in BPH/5 implantation sites, celecoxib (a selective Cox2 inhibitor) was administered at e6.5 (10mg/kg orally). This normalized IL-15 in BPH/5 e7.5 implantation sites compared to C57 veh-treated (n=4, p<.05). Flow cytometry confirmed a 2-fold increase in DBA+/CD122+ (dNK) cell numbers in BPH/5 e7.5 implantation sites from celecoxib vs veh-treated mice (16.6 vs 7.95). Moreover, the dNKs from e7.5 BPH/5 celecoxib-treated implantation sites had a 2-fold increase in IFNγ mean fluorescent intensity vs BPH/5 veh-treated. This demonstrates that uterine IL-15 overexpression in BPH/5 contributes to dNK loss and that normalization improves dNK activation by increasing their expression of IFNγ at the maternal-fetal interface. This highlights the significance of uterine inflammation in dNK regulation and suggests that aberrations in these cellular processes in early pregnancy may contribute to the placentopathies seen in PE pregnancies.