Avaliação da influência do estradiol sobre mecanismos da resposta imunológica em monócitos/macrófagos induzidos por Gardnerella vaginalis

Abstract

Gardnerella vaginalis is a commensal bacterium of the vaginal microbiota and the main agent of bacterial vaginosis (BV), a vaginal infection established due to an imbalance in this ecosystem. The pathogenesis of bacterial vaginosis is not well established; however, it is considered the main vaginal disorder that affects women of reproductive age, which may hypothesize the influence of female sex hormones in its pathogenesis. Objective: To evaluate the influence of 17β-estradiol (E2) on the immune response induced by Gardnerella vaginalis in in vitro models of female murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (hPMs) from women, with or without physiological ovarian activity. Methodology: in vivo and in vitro experiments were carried out. For the in vivo experiments in animals, the following were performed: body weight, uterine index and estradiol dosage; and in humans, laboratory tests such as blood count, biochemical and hormonal tests were performed. For the in vitro experiments, peritoneal macrophages from BALB/C sham surgery (SHAM) or ovariectomized (OVX) females and human peripheral blood monocytes (hPMs) from fertile and menopausal women were subdivided into three groups: cells inoculated with G. vaginalis, cells with sterile saline, both for 6 hours, and cells pretreated with 17β-Estradiol (E2), for 6 hours, and inoculated with G. vaginalis, also for 6 hours. Inflammatory markers such as: cytokines GM-CSF, IL-1β, TNF-α, IL-6, IL-12, IL-23, IL-27 and IL-10, total nitrites, hydrogen peroxide (H2O2), were measured in culture supernatant, and the expression of TLR-2, NF-kB and ERα and ERβ genes. In addition, a growth curve was performed in two groups. Group one, with only G. vaginalis and group two, G. vaginalis with 17β-estradiol, for a period of 24 hours. Results: For the analysis of the growth curve, the inhibitory activity of E2 on G. vaginalis can be verified. For animal experiments: In vivo, SHAM females had higher uterine weight and higher estradiol concentration, while OVX females had higher body weight. In vitro, the concentration of cytokines, IL-6, IL-8, IL-10 and inflammatory markers, total nitrites and (H2O2), showed high levels in MPMs of SHAM and OVX females inoculated with G. vaginalis when compared to G. vaginalis with sterile saline. When treated with E2, the MPMs of OVX females showed higher levels of IL-10 and hydrogen peroxide, when compared to untreated OVX. However, only the hydrogen peroxide matched the MPMs of the SHAM females. Similar results were observed for gene expressions of TLR-2, NF-kB and ERα and ERβ in the SHAM and OVX models between infected and sterile saline. Comparisons between infected cells from the SHAM, OVX and OVX treated with E2 models also showed higher levels of cytokines and inflammatory markers and gene expressions for cells from the SHAM model, with the exception only for the expression of ERβ. For the human experiments: In vivo, all selected research subjects were healthy, but a higher concentration of glucose, total cholesterol, LDL and TGP was observed in menopausal women and a higher concentration of leukocytes in women in their fertile period. For the serum dosage of sex hormones, it was observed that women in the fertile period had higher concentrations of 17β-estradiol and progesterone, while women in menopause had high concentrations of follicle-stimulating (FSH) and luteinizing (LH) hormones. In vitro, hPMs infected with G. vaginalis from women in the reproductive period and menopause showed higher levels of total nitrites, H2O2 and cytokines when compared to saline. However, infected hPMs from menopausal women had higher levels of H2O2 compared to hPMs from women in the reproductive period. With regard to cytokines, cells treated with E2 did not match those with endogenous E2, except for IL-10. Conclusion: In animal and human experiments, in relation to the influence of E2 on the acute inflammatory response to G. vaginalis, it is observed that the hormone has an immunomodulatory characteristic, but it cannot be exclusively defined as pro or anti-inflammatory. In this study, for endogenous E2, all cytokines, inflammatory markers and gene expressions showed higher levels, both in MPMs and hPMs. When treated with E2, MPMs from OVX females and hPMS from menopausal women showed similar response for IL-10 levels in relation to cells with endogenous E2, indicating an anti-inflammatory action. However, acute inflammation recognition and triggering genes also showed similarity between MPMs treated with E2 and cells with exogenous E2. Physiological influences, such as endogenous progesterone, may contribute for cells to present characteristics of an acute inflammatory response, and the treatment of cells, exclusively with E2, has not resulted in an equivalence of cell response with endogenous sex hormones. However, E2 showed a bacteriostatic characteristic in relation to G. vaginalis proliferation, and this hormone can modulate virulence factors of this microorganism, thus contributing to an absent or discrete immune response in the course of bacterial vaginosis.