Aspirin Inhibits the Inflammatory Response of Protease-Activated Receptor 1 in Pregnancy Neutrophils: Implications for Treating Women with Preeclampsia.

Neutrophils expressing cyclooxygenase-2 (COX-2) extensively infiltrate maternal blood vessels in preeclampsia, associated with vascular inflammation. Because pregnancy neutrophils also express protease-activated receptor 1 (PAR-1, F2R thrombin receptor), which they do not in non-pregnant subjects, they can be activated by proteases. We tested the hypothesis that aspirin at a dose sufficient to inhibit COX-2 would reduce inflammatory responses in preeclampsia neutrophils. Neutrophils were isolated from normal pregnant and preeclamptic women at approximately 30 weeks' gestation. Normal pregnancy neutrophils were treated with elastase, a protease elevated in preeclampsia, or elastase plus aspirin to inhibit COX-2, or elastase plus pinane thromboxane, a biologically active structural analog of thromboxane and a thromboxane synthase inhibitor. Preeclamptic pregnancy neutrophils were treated with the same doses of aspirin or pinane thromboxane. Confocal microscopy with immunofluorescence staining was used to determine the cellular localization of the p65 subunit of nuclear factor-kappa B (NF-κB) and media concentrations of thromboxane were measured to evaluate the inflammatory response. In untreated neutrophils of normal pregnant women, p65 was localized to the cytosol. Upon stimulation with elastase, p65 translocated from the cytosol to the nucleus coincident with increased thromboxane production. When neutrophils were co-treated with aspirin or pinane thromboxane, elastase was not able to cause nuclear translocation of p65 or increase thromboxane. In untreated neutrophils of preeclamptic women, the p65 subunit was present in the nucleus and thromboxane production was elevated, but when preeclamptic neutrophils were treated with aspirin or pinane thromboxane, p65 was cleared from the nucleus and returned to the cytosol along with decreased thromboxane production. These findings suggest that COX-2 is a downstream mediator of PAR-1 and demonstrate that PAR-1- mediated inflammation can be inhibited by aspirin. Given the extensive and ubiquitous expression of PAR-1 and COX-2 in preeclamptic women, consideration should be given to treating women with preeclampsia using a dose of aspirin sufficient to inhibit COX-2.

Gene Expression of Pregnancy Neutrophils Differs for Protease versus Lipopolysaccharide Stimulation.

Neutrophils, which extensively infiltrate maternal systemic blood vessels in preeclampsia, express protease-activated receptor 1 (PAR-1) but only during pregnancy. Neutrophils are generally considered to be non-specific in their response, but the pregnancy-specific expression of PAR-1 could result in a gene expression profile unique to pregnancy, which could help explain why the maternal inflammatory response in preeclampsia is systemic rather than localized. We sought to determine if gene expression of pregnancy neutrophils would differ if stimulated by a protease versus bacterial lipopolysaccharide (LPS). We isolated neutrophils from normal pregnant women at 30 weeks' gestation and cultured them with elastase or LPS. We used elastase because it is a protease elevated in women with preeclampsia, and it activates pregnancy neutrophils via PAR-1. RNA was isolated from the neutrophils for sequencing of the transcriptomes. We discovered many differences in the gene expression profiles. For example, exposure to elastase resulted in three times more uniquely expressed genes than LPS, and the number of significantly differentially upregulated and downregulated genes was greater for elastase. Analysis of canonical pathways revealed similarities for innate immunity but also differences. LPS treatment enriched more pathways, but elastase activated more genes in each pathway. Elastase treatment enriched the MAPK signaling pathway, whereas LPS did not. This is significant because MAPK is a key mediator of transcriptional responses. These findings indicate that protease stimulation of pregnancy neutrophils results in a different profile than stimulation with LPS, which may help explain why the sterile inflammatory response of preeclampsia is systemic and unique to pregnancy.

Epigenetic Regulation of Interleukin-17-Related Genes and Their Potential Roles in Neutrophil Vascular Infiltration in Preeclampsia.

DNA methylation is an epigenetic mechanism controlling gene expression, and reduced methylation is associated with increased gene expression. We hypothesized that IL-17 cytokines are regulated by DNA methylation, are elevated in the circulation of preeclamptic women, and stimulate vascular neutrophil chemokine expression, which could account for vascular infiltration of neutrophils in preeclampsia. We found significantly reduced DNA methylation of IL17A, IL17E, and IL17F genes in omental arteries of preeclamptic women, significantly reduced methylation of IL2, which regulates IL-17-producing T-lymphocytes, and significantly reduced methylation of genes encoding neutrophil chemokines and TNFα receptors related to lymphocyte function. Maternal plasma levels of IL-17A were significantly elevated in the second trimester of preeclamptic pregnancy as compared to normal pregnancy. To test if methylation regulates IL-17 cytokines, a lymphocyte cell line (Jurkat) was cultured with a hypomethylating agent. Hypomethylation increased expression of IL17E (aka IL25), IL17F, and IL2. IL17A was not expressed by Jurkat cells. To test the potential role of IL-17 cytokines in vascular neutrophil infiltration associated with preeclampsia, human vascular smooth muscle cells were cultured with IL-17 cytokines. IL-17A, but not IL-17E or IL-17F, increased gene expression of neutrophil chemokines (IL-8, CXCL5, and CXCL6) that are increased in vascular smooth muscle of preeclamptic women. The monocyte chemokine, CCL-2, was not increased. TNFα also increased neutrophil chemokines. IL-17 cytokines are regulated by DNA methylation; IL-17A is elevated in preeclampsia and stimulates expression of neutrophil chemokines in vascular smooth muscle. IL-17A could be responsible for vascular infiltration of neutrophils in preeclampsia.

Patterns of Maternal Neutrophil Gene Expression at 30 Weeks of Gestation, but Not DNA Methylation, Distinguish Mild from Severe Preeclampsia.

Neutrophils are activated and extensively infiltrate blood vessels in preeclamptic women. To identify genes that contribute to neutrophil activation and infiltration, we analyzed the transcriptomes of circulating neutrophils from normal pregnant and preeclamptic women. Neutrophils were collected at 30 weeks' gestation and RNA and DNA were isolated for RNA sequencing and 5-hydroxy-methylcytosine (5-hmC) sequencing as an index of dynamic changes in neutrophil DNA methylation. Women with normal pregnancy who went on to develop mild preeclampsia at term had the most uniquely expressed genes (697) with 325 gene ontology pathways upregulated, many related to neutrophil activation and function. Women with severe preeclampsia who delivered prematurely had few pathways up- or downregulated. Cluster analysis revealed that gene expression in women with severe preeclampsia was an inverse mirror image of gene expression in normal pregnancy, while gene expression in women who developed mild preeclampsia was remarkably different from both. DNA methylation marks, key regulators of gene expression, are removed by the action of ten-eleven translocation (TET) enzymes, which oxidize 5-methylcytosines (5mCs), resulting in locus-specific reversal of DNA methylation. DNA sequencing for 5-hmC revealed no differences among the three groups. Genome-wide DNA methylation revealed extremely low levels in circulating neutrophils suggesting they are de-methylated. Collectively, these data demonstrate that neutrophil gene expression profiles can distinguish different preeclampsia phenotypes, and in the case of mild preeclampsia, alterations in gene expression occur well before clinical symptoms emerge. These findings serve as a foundation for further evaluation of neutrophil transcriptomes as biomarkers of preeclampsia phenotypes. Changes in DNA methylation in circulating neutrophils do not appear to mediate differential patterns of gene expression in either mild or severe preeclampsia.

Proteases Activate Pregnancy Neutrophils by a Protease-Activated Receptor 1 Pathway: Epigenetic Implications for Preeclampsia.

We tested a novel hypothesis that elevated levels of proteases in the maternal circulation of preeclamptic women activate neutrophils due to their pregnancy-specific expression of protease-activated receptor 1 (PAR-1). Plasma was collected longitudinally from normal pregnant and preeclamptic women and analyzed for MMP-1 and neutrophil elastase. Neutrophils were isolated for culture and confocal microscopy. Omental fat was collected for immunohistochemistry. Circulating proteases were significantly elevated in preeclampsia. Confocal microscopy revealed that tet methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, and p65 subunit of NF-κB were strongly localized to the nucleus of untreated neutrophils of preeclamptic women, but in untreated neutrophils of normal pregnant women they were restricted to the cytosol. Treatment of normal pregnancy neutrophils with proteases activated PAR-1, leading to activation of RhoA kinase (ROCK), which triggered translocation of TET2 and p65 from the cytosol into the nucleus, mimicking the nuclear localization in neutrophils of preeclamptic women. IL-8, an NF-κB-regulated gene, increased in association with TET2 and p65 nuclear localization. Co-treatment with inhibitors of PAR-1 or ROCK prevented nuclear translocation and IL-8 did not increase. Treatment of preeclamptic pregnancy neutrophils with inhibitors emptied the nucleus of TET2 and p65, mimicking the cytosolic localization of normal pregnancy neutrophils. Expression of PAR-1 and TET2 were markedly increased in omental fat vessels and neutrophils of preeclamptic women. We conclude that elevated levels of circulating proteases in preeclamptic women activate neutrophils due to their pregnancy-specific expression of PAR-1 and speculate that TET2 DNA de-methylation plays a role in the inflammatory response.

Placental Production of Eicosanoids and Sphingolipids in Women Who Developed Preeclampsia on Low-Dose Aspirin.

Low-dose aspirin, which selectively inhibits thromboxane synthesis, is now standard of care for the prevention of preeclampsia in at risk women, but some women still develop preeclampsia despite an aspirin regimen. To explore the "aspirin failures," we undertook a comprehensive evaluation of placental lipids to determine if abnormalities in non-aspirin sensitive lipids might help explain why some women on low-dose aspirin develop preeclampsia. We studied placentas from women with normal pregnancies and women with preeclampsia. Placental villous explants were cultured and media analyzed by mass spectrometry for aspirin-sensitive and non-aspirin-sensitive lipids. In women who developed severe preeclampsia and delivered preterm, there were significant elevations in non-aspirin-sensitive lipids with biologic actions that could cause preeclampsia. There were significant increases in 15- and 20-hydroxyeicosatetraenoic acids and sphingolipids: D-e-C18:0 ceramide, D-e-C18:0 sphingomyelin, D-e-sphingosine-1-phosphate, and D-e-sphinganine-1-phosphate. With regard to lipids sensitive to aspirin, there was no difference in placental production of thromboxane or prostacyclin, but prostaglandins were lower. There was no difference for isoprostanes, but surprisingly, anti-inflammatory omega 3 and 6 PUFAs were increased. In total, 10 of 30 eicosanoids and 5 of 42 sphingolipids were abnormal in women with severe early onset preeclampsia. Lipid changes in women with mild preeclampsia who delivered at term were of lesser magnitude with few significant differences. The placenta produces many aspirin-sensitive and non-aspirin-sensitive lipids. Abnormalities in eicosanoids and sphingolipids not sensitive to aspirin might explain why some aspirin-treated women develop preeclampsia.