Investigating the effects of sex hormone supplementation on bone marrow-derived macrophage polarization and pro-atherogenic function

Abstract

Atherosclerosis, the main underlying cause of most cardiovascular disease (CVD), is characterized by progressive inflammation of the vascular intima. Macrophages are inflammatory cells centrally involved in atherogenesis. Macrophage polarization into pro-inflammatory or anti-inflammatory phenotypes has a significant impact on disease progression. Sex differences in the development and progression of CVD are well established, but the effects of sex hormones on macrophage polarization and pro-atherogenic functions are not well described. We hypothesized that sex hormones directly modulate macrophage polarization and pro-atherogenic function, and thereby regulate the progression of atherosclerosis. Bone marrow-derived monocytes from adult male and female C57BL/6 mice were differentiated into macrophages using macrophage colony-stimulating factor (20 ng/mL) and pre-treated with either 17B-estradiol (100 nM), testosterone (100 nM), or a vehicle control for 24 hours. Pre-treated macrophages were polarized into the pro-inflammatory or anti-inflammatory phenotypes and the effects of sex hormone supplementation on gene expression of macrophage phenotypic markers were assessed using RT-qPCR. Protein concentration of inflammatory markers including IL-1B was quantified using an addressable laser bead immunoassay. A Transwell migration assay was employed to determine changes in macrophage migration, and changes in lipid accumulation were determined by cell staining and fluorescence microscopy. Sex differences were observed in macrophage polarization, inflammatory responses, migration, and lipid accumulation. Pre-treatment with 17B-estradiol significantly impaired gene expression of pro-inflammatory macrophage markers and production of IL-1B in pro-inflammatory macrophages. In anti-inflammatory macrophages, 17B-estradiol significantly upregulated expression of anti-inflammatory markers and enhanced cell migration. Pre-treatment with testosterone enhanced anti-inflammatory marker mRNA expression in anti-inflammatory macrophages and impaired production of IL-1B by pro-inflammatory macrophages. Our observations suggest a protective role of 17B-estradiol and testosterone in atherogenesis and may contribute to the sexual dimorphisms in CVD observed in human patients.