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

Abstract

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.

Keywords: Chemokines; DNA methylation; Interleukin-17; Neutrophils; Preeclampsia; TNFα.

1.

DNA methylation in omental arteries of women with normal pregnancy and women with preeclampsia. DNA methylation of IL17A, IL17E, and IL17F were all significantly lower in women with preeclampsia as compared to women with normal pregnancy. Methylation of IL2 was also significantly lower in women with preeclampsia. NP, normal pregnancy (n = 5); PE, preeclampsia (n = 7); * P < 0.05, ** P < 0.01

2.

Maternal plasma levels of IL-17A in women with normal pregnancy (NP) and women who developed preeclampsia (PE). Samples were collected longitudinally throughout pregnancy. Women with preeclampsia had significantly elevated levels of IL-17A in the second trimester of pregnancy as compared to women with normal pregnancy (weeks 13 – 28), but not in the third trimester (weeks 29 – 40). ** P < 0.01 (n = 28 for NP subjects and n = 35 for PE subjects)

3.

Regulation of IL17 and IL2 genes by DNA methylation. 5-Aza-induced DNA hypomethylation increased expression of IL17E, IL17F and IL2 genes in Jurkat cells demonstrating that these genes are epigenetically regulated. IL17A was not expressed by Jurkat cells. n = 9, **P < 0.01, ****P < 0.0001

4.

Regulation of vascular smooth muscle chemokines by IL-17 cytokines. IL-17A increased gene expression of neutrophil chemokines, IL8, CXCL5 and CXCL6, but not of the monocyte chemokine, CCL2, in vascular smooth muscle cells. IL-17E and IL-17F did not produce significant changes in neutrophil or monocyte chemokine gene expression in vascular smooth muscle cells. n = 12, ***P < 0.001

5.

Regulation of vascular smooth muscle chemokines by TNFα. TNFα increased gene expression of neutrophil chemokines, IL8, CXCL5 and CXCL6, in vascular smooth muscle cells. n = 6, **P < 0.01, ****P < 0.0001

6.

Representative images of immunohistochemical staining for CXCL 5&6 in omental fat vessels of women with normal pregnancy (NP) and women with preeclampsia (PE). Little staining was present in vessels of women with normal pregnancy (Panel A) as compared to intense dark brown staining in vessels of women with preeclampsia (Panel B). Panels C and D show examples of the areas of staining highlighted in yellow between groups. The majority of staining for CXCL 5&6 was in the vascular smooth muscle. Panel E is a negative control. Panel F shows the quantification of the area of staining. The area of staining was significantly greater in PE (n = 3) than in NP (n = 3). Pictures were taken with a 40X lens. VL, Vessel lumen, VSM, vascular smooth muscle, A, adipose cell, * P < 0.05.