The influence of 17-β-estradiol on bioenergetic and estrogen metabolism in macrophages

Abstract

Breast cancer is a multifactorial disease influenced by a variety of factors in the tumour microenvironment (TME), including hormones such as estrogen, and immune cells such as macrophages. Imbalances in estrogen metabolism and certain estrogen metabolites have been linked to breast cancer. Estrogen has also been shown to influence macrophage polarization. The metabolic and functional plasticity of macrophages allow these cells to play a dual role in breast cancer by either inhibiting or promoting tumourigenesis based on their activation (polarization) state. Considering the potential role of both estrogen and macrophages in breast cancer, it is possible that interaction between the two can further affect tumour dynamics. Macrophage polarization is tightly linked to metabolic state, however it is not known how estrogens affect the bio-energetic metabolism of naïve, pro-, or anti-inflammatory macrophages. Furthermore, estrogen metabolism in macrophages is not well studied. In this study the effect of 17-β-estradiol (E2) on macrophage bio-energetics was compared to that of lipopolysaccharide (LPS) and interleukin-4 (IL-4) at different glucose concentrations (5 mM, 11 mM, and 25 mM) using the Seahorse Extracellular Flux Analyzer to measure mitochondrial oxidative phosphorylation (OXPHOS). After identifying the most optimal glucose concentration from these and additional glucose consumption assays, the effect of E2 on the bio-energetic metabolism of pro- and anti-inflammatory RAW 264.7 cells and mouse bonemarrow derived macrophages (BMDMs) was also investigated. Finally, cell culture medium from RAW 264.7 cells (incubated with or without LPS, IL-4, and E2) was collected to analyse estrogen metabolite production with LCMS/ MS. Bio-energetics results reveal that the effect of LPS, IL-4, and E2 on macrophage metabolism is influenced by the glucose concentration in the culture medium. E2 induced an oxidative metabolism in macrophages, similar to IL-4 treatment. Moreover, E2 caused a shift in the bio-energetic metabolism of LPS-treated (pro-inflammatory) cells towards a metabolic profile comparable to naïve cells. Since macrophage polarization and metabolism is reciprocally coupled, this may mean that E2 present in the breast tissue may dampen or limit pro-inflammatory, anti-cancer macrophages (present during the early stages of tumor development) by inducing a metabolic switch. This may favor tumor initiation and progression. LC-MS/MS results showed that macrophages can synthesise and secrete estradiol (particularly the less active 17-α-estradiol) into their environment, where it may have the potential to affect the functionality of other neighboring cells in the TME. Furthermore, LPS-treated macrophages seemed to produce more 17-α-estradiol and 17-β-estradiol compared to anti-inflammatory cells. This may be due to the involvement of LPS in aromatase modulation. The findings from this study suggest that the interplay between estrogen and macrophages (which can also be influenced by other factors such as glucose concentration) may alter tumour dynamics, but probably not via the production of carcinogenic estrogen metabolites by macrophages.