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Estradiol inhibits ongoing autoimmune neuroinflammation and NFkappaB-dependent CCL2 expression in reactive astrocytes - PubMed

  • ️Fri Jan 01 2010

Estradiol inhibits ongoing autoimmune neuroinflammation and NFkappaB-dependent CCL2 expression in reactive astrocytes

Sébastien N Giraud et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Astroglial reactivity associated with increased production of NFkappaB-dependent proinflammatory molecules is an important component of the pathophysiology of chronic neurological disorders such as multiple sclerosis (MS). The use of estrogens as potential anti-inflammatory and neuroprotective drugs is a matter of debate. Using mouse experimental allergic encephalomyelitis (EAE) as a model of chronic neuroinflammation, we report that implants reproducing pregnancy levels of 17beta-estradiol (E2) alleviate ongoing disease and decrease astrocytic production of CCL2, a proinflammatory chemokine that drives the local recruitment of inflammatory myeloid cells. Immunohistochemistry and confocal imaging reveal that, in spinal cord white matter EAE lesions, reactive astrocytes express estrogen receptor (ER)alpha (and to a lesser extent ERbeta) with a preferential nuclear localization, whereas other cells including infiltrated leukocytes express ERs only in their membranes or cytosol. In cultured rodent astrocytes, E2 or an ERalpha agonist, but not an ERbeta agonist, inhibits TNFalpha-induced CCL2 expression at nanomolar concentrations, and the ER antagonist ICI 182,170 blocks this effect. We show that this anti-inflammatory action is not associated with inhibition of NFkappaB nuclear translocation but rather involves direct repression of NFkappaB-dependent transcription. Chromatin immunoprecipitation assays further indicate that estrogen suppresses TNFalpha-induced NFkappaB recruitment to the CCL2 enhancer. These data uncover reactive astrocytes as an important target for nuclear ERalpha inhibitory action on chemokine expression and suggest that targeting astrocytic nuclear NFkappaB activation with estrogen receptor alpha modulators may improve therapies of chronic neurodegenerative disorders involving astroglial neuroinflammation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Estradiol treatment after disease onset suppresses the clinical symptoms of experimental autoimmune encephalomyelitis. Clinical score is shown of placebo- (EAE, solid circles, n = 14) and estradiol (EAE + E2, open squares, n = 13) -treated EAE mice. Implants (placebo or E2, 5 mg) were performed at day 14 postimmunization (arrow).

Fig. 2.
Fig. 2.

Treatment with estradiol in vivo reduces CCL2 expression in the spinal cord of EAE mice. (A) CCL2 immunoreactivity (IR) and corresponding DAPI staining on hemisections of control, EAE, and E2-treated EAE spinal cords. Increased CCL2-IR in EAE mice correlated with multifocal areas of increased infiltrated cells disseminated in the white matter (as revealed by DAPI staining, arrowheads). In E2-treated EAE mice, CCL2-IR was decreased in the white matter, remaining in smaller areas of infiltrating cells restricted around leptomeninges. For pictures of whole hemisections from ×5 objective, corner areas outside the spinal cord have been filled because of rotation of the initial pictures and presence of occasional nerve remainings. (B) Confocal imaging showing colocalization of CCL2-IR (green) in astrocytic fibers (GFAP-IR, red). Each panel is a z-stack of eight consecutive confocal sections with 1-μm increments. (Scale bars, 10 μm.) (C) qPCR of CCL2 mRNA from spinal cord extracts. Difference between placebo- and E2-treated EAE mice (post hoc analysis): ***, P < 0.001.

Fig. 3.
Fig. 3.

Increased estrogen receptor ERα and ERβ immunoreactivities in EAE white matter. (A, D, G, and J) Control mouse; (B, E, H, and K) EAE mouse; (C, F, I, and L) E2-treated EAE mouse. (A–F) ERα immunoreactivity (C1335 antibody, A–C) or ERβ immunoreactivity (D–F) in hemisections of the spinal cord. Arrowheads point to increased immunoreactivities (IR) in areas of white matter infiltrates in EAE mice. (Scale bar, 200 μm.) (G–I) Higher magnification of ERα labeling (C1355 antibody) in the ventral funiculus with (J–L) corresponding DAPI staining. (Scale bar, 80 μm.)

Fig. 4.
Fig. 4.

Confocal imaging of estrogen receptors in the spinal cord of control and EAE mice. (A–C) Radial glia (arrowhead) stained with anti-ERα C1355 (A), anti-GFAP and DAPI (B), or anti-ERα C1355 and anti-GFAP (C) in the white matter of a control mouse. (D–F) Radial glia (arrowhead) stained with anti-ERα clone 60C (D), anti-GFAP and DAPI (E), or anti-ERα and anti-GFAP (F) in the white matter of a control mouse. (G–I) Example of reactive astrocytes stained with anti-ERα clone 60C (G), anti-GFAP and DAPI (H), or anti-ERα and anti-GFAP (I) in EAE white matter. (K–P) Radial glia (arrowhead) and reactive astrocytes (arrows) stained with anti-ERβ (K and N), anti-GFAP and DAPI (L and O), or anti-ERβ and anti-GFAP (M and P) in EAE white matter. (Q and R) Higher magnification showing nuclear localization of ERα (Q, clone 60C) or ERβ (R) in GFAP-immunoreactive cells and corresponding DAPI staining (Q′ and R′). (S) Dorsal horn neurons stained with anti-ERα (clone 60C, green) and DAPI. (T) Motoneuron stained with anti-ERβ and DAPI (the contrasts for the green and blue channels have been enhanced by ×2 and ×3, respectively, for better visualization). (U and U′) CD45-IR cells in a perivascular infiltrate with ERβ localization restricted to the membrane or cytosol compartment (arrowheads); the arrow points to an endothelial cell identified by its long fusiform nucleus and constituting part of a blood vessel when observed in consecutive sections. In A–P, each image is a z-stack of eight consecutive confocal sections with 1-μm increments. (Scale bar, 25 μm.) In Q–U, single confocal sections are shown. (Scale bar, 13 μm.)

Fig. 5.
Fig. 5.

Effects of cytokines and estradiol on CCL2 expression in astrocyte cultures from neonatal rat cortex. (A) Effect of proinflammatory cytokines (50 ng/mL) on CCL2 content in medium. Data are expressed as content ratio (n = 6–9/group) relative to control (3.8 ng/mL). Con, control; E2, 17β-estradiol; IFN, IFNγ; IL-1, interleukin-1β; TNF, TNFα. ANOVA: F4,26 = 20.8, P < 0.001. (B and C) Effect of E2 (10 nM) and TNFα (10 ng/mL) on (B) CCL2 mRNA expression (F3,16 = 10.34, P < 0.0001, n = 4–6/group, two experiments), (C) CCL2 content in the medium (n = 5–9/group; ANOVA, F3,27 = 10.34, P < 0.0001; control levels = 3.9 ng/mL). (D) Semiquantitative analysis of CCL2 immunohistofluorescence (IHF) in astrocytes treated with E2 and TNFα. Data are expressed as ratio of immunofluorescence intensity relative to control (n = 3/group). (E and F) Dose-dependent effect of E2 on (E) CCL2 mRNA or (F) CCL2 levels in the medium. Data are expressed as relative ratio compared to controls (two dishes/dose/group). Post hoc analysis: (A–D) Difference vs. control, *, P < 0.05; **, P < 0.01; ***, P < 0.001; difference vs. TNFα, #, P < 0.05; (E and F) difference vs. TNFα without estradiol, *, P < 0.05; **, P < 0.01.

Fig. 6.
Fig. 6.

Estradiol does not impede TNFα-induced p65 nuclear translocation but suppresses NFκB-dependent transcription in cultured astrocytes. (A) ERα and ERβ immunolocalization in mouse spinal cord astrocyte cultures. ERα is detected in the nucleus as well as in the cytoplasm/membrane compartments whereas ERβ is detected mainly in the cytoplasm. GFAP (red) and DAPI (blue) stainings and corresponding ERα and ERβ immunoreactivities (green) are shown. (scale bar, 25 μm.) (B) p65 immunoreactivity (green) in astrocytes stained for GFAP (red) and DAPI (blue). (scale bar, 100 μm.) a and a′, controls; b and b′, E2, 10 nM; c and c′, TNFα, 10 ng/mL; and d and d′, TNFα + E2. (C) NFκB-dependent transcription assay. Mouse neonatal spinal cord astrocytes (NSCA) transfected with a luciferase NFκB reporter plasmid were treated with vehicle (Con) or 10 nM E2 for 30 min, followed by 2 h incubation in the presence or absence of 10 ng/mL TNFα, before luciferase assay. Data are expressed as relative light units (n = 3/group). Post hoc analysis: **, P < 0.01 vs. control. (D) Recruitment of p65 to the NFκB-dependent CCL2 enhancer in NSCA shown by ChIP assay. Cells were treated with E2 or TNFα as in C. Fragmented chromatin was subjected to ChIP analysis and real-time PCR. Left, percentage of CCL2 enhancer precipitated with anti-p65 relative to corresponding input (n = 3/group). Post hoc analysis: **, P < 0.01 vs. control. Right, representative melting temperature (Tm) curves showing recovery of CCL2 amplicon from anti-p65 ChIP and 0.5% input samples (Tm 82 °C) but not from mock sample (immunoprecipitation with a rabbit IgG).

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References

    1. Zamvil SS, Steinman L. Diverse targets for intervention during inflammatory and neurodegenerative phases of multiple sclerosis. Neuron. 2003;38:685–688. - PubMed
    1. Luo J, Ho P, Steinman L, Wyss-Coray T. Bioluminescence in vivo imaging of autoimmune encephalomyelitis predicts disease. J Neuroinflammation. 2008;5:6. - PMC - PubMed
    1. Barres BA. The mystery and magic of glia: A perspective on their roles in health and disease. Neuron. 2008;60:430–440. - PubMed
    1. Brambilla R, et al. Transgenic inhibition of astroglial NF-kappa B improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation. J Immunol. 2009;182:2628–2640. - PMC - PubMed
    1. McKay LI, Cidlowski JA. Molecular control of immune/inflammatory responses: Interactions between nuclear factor-kappa B and steroid receptor-signaling pathways. Endocr Rev. 1999;20:435–459. - PubMed

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