Effects of 17β-Estradiol on Monocyte/Macrophage Response to Staphylococcus aureus: An In Vitro Study

Abstract

To describe how 17β-estradiol (E2) influence in the monocyte/macrophage response induced by S. aureus in in vitro models of murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (HPBM). MPMs (2 x 10⁵/ml) were isolated from sham (n=3) and ovariectomized (OVX) females (n = 3) and males (n = 3) after induction by thioglycolate. The MPMs obtained from OVX females and males were treated for 24 hours with 17β-estradiol (E2) (10^-7 M), and after that, inoculation with S. aureus was carried out for 6 hours. The macrophages were collected and destined to evaluate the relative gene expression of TNF-α, IL-1β, IL-6, IL-8 and TLR2. For the in vitro model of HPBMs, six men and six women of childbearing age were selected and HPBMs were isolated from samples of the volunteers' peripheral blood. In women, blood was collected both during menstruation and in the periovulatory period. HPBMs were inoculated with S. aureus for 6 hours and the supernatant was collected for analysis of cytokines by Luminex and the HPBMs were removed for analysis of 84 genes involved in the host's response to bacterial infections by RT-PCR array. Previous treatment with E2 decreased the gene expression and production of proinflammatory cytokines, such as TNF-α, IL-1β and IL-6 and decreased the expression of TLR2 tanto em MPMs quanto em HPBMs. The analysis of gene expression shows that E2 inhibited the NFκB pathway. It is suggested that 17β-estradiol acts as an immunoprotective in the monocyte/macrophage response induced by S. aureus.

Keywords: Staphylococcus aureus; immune response; immunomodulation; macrophage; sex steroids.

Figure 1

(1) Relative gene expression of the cytokines TNF (A1), IL-1 (B1), IL-6 (C1) and IL-8 (D1) of MPMs (2 x 10⁵/ml) from sham (n = 6) and OVX females (n = 6) inoculated with S. aureus (10⁸ CFU) for 6 h. (2) Relative gene expression of the cytokines TNF (A2), IL-1 (B2), IL-6 (C2) and IL-8 (D2) of MPMs (2 x 10⁵/ml) from OVX females (n = 6) stimulated or not with 17β-estradiol (E2) (10^-7 M) for 24 h before inoculation with S. aureus (10⁸ CFU) for 6 h. (3) Relative gene expression of cytokines TNF (A3), IL-1 (B3), IL-6 (C3) and IL-8 (D3) of MPMs (2 x 10⁵/ml) of males (n = 6) stimulated or not with 17β-estradiol (E2) (10^-7 M) for 24 h before inoculated with S. aureus (10⁸ CFU) for 6 h. Evaluated by RT-qPCR array. Data expressed as mean ± SDM. *P < 0.05. **P < 0.01.

Figure 2

(A) Relative gene expression of TLR2 of MPMs (2 x 10⁵/ml) from sham (n = 6) and OVX females inoculated with S. aureus (10⁸ CFU) for 6 h. (B) Relative gene expression of TLR2 from OVX females (n = 6) stimulated or not with 17b-estradiol (E2) (10^-7 M) for 24 h before inoculation with S. aureus (10⁸ CFU) for 6 h. (C) Relative gene expression of TLR2 of MPMs (2 x 10⁵/ml) of males (n = 6) stimulated or not with 17b-estradiol (E2) (10^-7 M) for 24 h before inoculation with S. aureus (10⁸ CFU) for 6 h. Evaluated by RT-qPCR array. Data expressed as mean ± SDM. *P < 0.05.

Figure 3

Concentration of IL-1β, IL-6, TNF-α, GM-CSF, IL-10, IL-12, IL-23 and IL-27 in the supernatant of HPBMs of men and women inoculated with S. aureus (10⁸ CFU). HPBMs of women in the first two days of their menstrual period or in the periovulatory period inoculated with S. aureus (A1–A8); HPBMs of men or women in the periovulatory period inoculated with S. aureus (B1–B8); HPBMs from men inoculated with S. aureus or men inoculated with S. aureus and previously treated with 17β-estradiol (E2) (10^-7 M) (C1–C8). Evaluated by Luminex. Data expressed as mean ± SDM. *P < 0.05. **P < 0.01.

Figure 4

(A) Analysis of 84 genes involved in the host’s response to bacterial infections of HPBMs inoculated with S. aureus from women during their menstrual period compared to HPBMs from women during their fertile period. Analysis performed with the Human Innate & Adaptive Immune Responses PCR Array kit (Qiagen - SABioscience). Positively regulated genes (green) and negatively regulated genes (red). *P < 0.05 vs. HPBMs inoculated with S. aureus from women during their fertile period. (B) Analysis of 84 genes involved in the host’s response to bacterial infections of HPBMs inoculated with S. aureus from women during their fertile period compared to HPBMs from men. Analysis performed with the Human Innate & Adaptive Immune Responses PCR Array kit (Qiagen - SABioscience). Negatively regulated genes (red). *P < 0.05 vs. HPBMs inoculated with S. aureus from men. (C) Analysis of 84 genes involved in the host’s response to bacterial infections of HPBMs inoculated with S. aureus from men treated with E2 compared to HPBMs from men. Analyzed by the Human Innate & Adaptive Immune Responses PCR Array kit (Qiagen - SABioscience). Negatively regulated genes (red). Data expressed as mean ± SDM. *P < 0.05 vs. HPBMs inoculated with S. aureus from men.