Estrogen treatment decreases matrix metalloproteinase (MMP)-9 in autoimmune demyelinating disease through estrogen receptor alpha (ERalpha)

Abstract

Matrix metalloproteinases (MMPs) have a crucial function in migration of inflammatory cells into the central nervous system (CNS). Levels of MMP-9 are elevated in multiple sclerosis (MS) and predict the occurrence of new active lesions on magnetic resonance imaging (MRI). This translational study aims to determine whether in vivo treatment with the pregnancy hormone estriol affects MMP-9 levels from immune cells in patients with MS and mice with experimental autoimmune encephalomyelitis (EAE). Peripheral blood mononuclear cells (PBMCs) collected from three female MS patients treated with estriol and splenocytes from EAE mice treated with estriol, estrogen receptor (ER) alpha ligand, ERbeta ligand or vehicle were stimulated ex vivo and analyzed for levels of MMP-9. Markers of CNS infiltration were assessed using MRI in patients and immunohistochemistry in mice. Supernatants from PBMCs obtained during estriol treatment in female MS patients showed significantly decreased MMP-9 compared with pretreatment. Decreases in MMP-9 coincided with a decrease in enhancing lesion volume on MRI. Estriol treatment of mice with EAE reduced MMP-9 in supernatants from autoantigen-stimulated splenocytes, coinciding with decreased CNS infiltration by T cells and monocytes. Experiments with selective ER ligands showed that this effect was mediated through ERalpha. In conclusion, estriol acting through ERalpha to reduce MMP-9 from immune cells is one mechanism potentially underlying the estriol-mediated reduction in enhancing lesions in MS and inflammatory lesions in EAE.

Figure 1. MMP-9 regulation by estriol in multiple sclerosis

Six month of estriol treatment reduced MMP-9 bioactivity in peripheral blood mononuclear cell (PBMC) culture supernatants (PHA stimulated) as measured by zymography compared to pre-treatment (pre) and 3 month after treatment cessation (post) in three female patients with relapsing-remitting MS (A). Estriol significantly decreased MMP-9/TIMP1 ratio (B). This effect was driven by decreases in MMP-9 levels (C), while estriol had no effect on TIMP1 (D). No significant changes were observed on MMP-2/TIMP2 ratio (E), MMP-2 (F), or TIMP2 (G). Decreased volumes of gadolinium-enhancing lesions on MRI occurred during estriol treatment (H). Representative scans from one subject show resolution of an enhancing lesion (I, white arrow). Intracellular staining using flow cytometry indicated that T cells (upper right quadrant) were a major source of MMP-9 in PHA activated PBMCs (J).

Figure 2. Estriol regulation of MMP-9 in active experimental autoimmune encephalomyelitis (EAE)

Autoantigen-stimulated splenocyte cultured supernatants from estriol treated EAE mice showed decreased MMP-9 bioactivity (A, quantification in panel B) as measured by zymography. Band intensity measurements were normalized by setting the mean band intensity of the vehicle treated group as 100%. Values are expressed as relative intensity (%) for all animals. Estriol treated animals also showed significantly reduced MMP-9 protein levels (C) compared to vehicle treated (n=5 in each group). This was accompanied by abrogation of clinical disease (D). Estriol treatment decreased infiltration into the CNS by T cells (E). Representative images are shown in panels F–G (T cells are labeled with anti-CD3 antibodies, green staining). Similarly, estriol treatment decreased CNS infiltration by macrophages (E). Representative images are shown in panels H–I (macrophages are labeled with anti-Mac3 Gold antibodies, green staining). The nuclear stain DAPI (pseudocolored red) was used to identify all cell nuclei. Images show dorsal column of spinal cord at ×10 magnification (insets at ×40).

Figure 3. Estriol-induced MMP-9 downregulation in EAE is mediated through ERα

Decreased bioactivity of MMP-9 in supernatants from splenocyte cultures compared to vehicle treated was seen in ERα ligand and estriol treated mice, but not in vehicle or ERβ ligand treated mice (A pooled supernatants from 4 animals in each group, B supernatants from individual animals, C quantification). Band intensity measurements were normalized by setting the mean band intensity of the vehicle treated group as 100%. Values are expressed as relative intensity (%) for all animals. Significantly decreased MMP-9 protein levels were observed in ERα ligand treated EAE mice (n=5) compared to vehicle treated EAE (n=4), while there was no difference between vehicle treated and ERβ ligand (n=3) treated mice (D). ERα treatment completely abrogated clinical disease (E). ERα, but not ERβ, ligand treatment reduced infiltration into the CNS by T cells (F). Representative images are shown in panels G–H (T cells are labeled with anti-CD3 antibodies, green staining). Similarly, ERα, but not ERβ, ligand treatment decreased macrophages infiltration (F). Representative images are shown in panels I–J (macrophages are labeled with anti-Mac3 antibodies, green staining). The nuclear stain DAPI (pseudocolored red) was used to identify all cell nuclei. Images show dorsal column of spinal cord at ×10 magnification (insets at ×40).