Avaliação do efeito de derivados lipofílicos da genisteína na ativação de macrófagos in vitro e na modulação da resposta imune no modelo de EAE

Abstract

Multiple Sclerosis (MS) is a severe and disabling chronic autoimmune inflammatory demyelinating disease of the central nervous system (CNS), that affect around one million people at entire world. Among the therapeutic agents currently approved for the treatment of MS can be cited the interferon beta (IFN-β) and the glatiramer acetate, however, these agents do not promote the cure or full recovery of patients in advanced stages. Several studies showed that hormones, such as estradiol and progesterone, could reduce the severity of experimental autoimmune encephalomyelitis (EAE). The isoflavones share many structural similarities with endogenous estrogens. Studies with genistein showed the beneficial effects of isoflavones in EAE. However, the chemical structure of genistein has characteristics that limit its clinical application, such as rapid excretion and decreased serum levels. This study aimed evaluate the effect of seven lipophilic derivatives of genistein in the modulation of in vitro response of murine J774A.1 macrophages activated with lipopolysaccharide and IFN-g, and verify the performance of one in vitro selected analogue on modulating in vivo the immune response in EAE. The lipophilic derivatives were evaluated in LPS+IFN-g-activated J774A.1 macrophage cultures to their effects on cytotoxicity and nitric oxide, IL-12 and TNF-α production. The effects of the selected genistein derivative 3 (7-O-genistein-tetradecanoil - TDG) on the development of EAE induced in C57BL/6 mice immunized with MOG35-55 were performed. At 14th day after induction, mice were treated with TDG for seven days. At 21st day the percentage of mononuclear cells isolated from brains expressing CD4+, CD3+, CTLA-4+ and Foxp3+ and producing IL-17+ and IL-10+ were determined by flow cytometry. The concentration of IL-6, IFN-g and IL-10 in brain and spinal cord were determined by ELISA. Histological analysis of brain and spinal cord were performed by hematoxylin and eosin staining. The results showed that the modification of genistein enables the generation of non-cytotoxic compounds with increased IL-12 inhibition, despite of failed TNF-α inhibition. The NO production was inhibited by the monoester (2,3) and monoether (6,7) compounds in a dose-dependent manner. The in vivo data indicated that treatment with TDG improved the clinical signs of EAE which can be correlated with a reduction in the percentage of cells producing IL-17 and the increment of Foxp3+CD4+ cell population at brain. Furthermore, treatment with TDG increased the release of IL-10 and expression of CTLA-4 and reduced the release of IFN-g and IL-6. Altogether, the data suggest an immunomodulatory role of the TDG in EAE and possibly MS.