Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis is an inflammatory, neurodegenerative disease for which experimental autoimmune encephalomyelitis (EAE) is a model. Treatments with estrogens have been shown to decrease the severity of EAE through anti-inflammatory mechanisms. Here we investigated whether treatment with an estrogen receptor alpha (ERalpha) ligand could recapitulate the estrogen-mediated protection in clinical EAE. We then went on to examine both anti-inflammatory and neuroprotective mechanisms. EAE was induced in wild-type, ERalpha-, or ERbeta-deficient mice, and each was treated with the highly selective ERalpha agonist, propyl pyrazole triol, to determine the effect on clinical outcomes, as well as on inflammatory and neurodegenerative changes. ERalpha ligand treatment ameliorated clinical disease in both wild-type and ERbeta knock-out mice, but not in ERalpha knock-out mice, thereby demonstrating that the ERalpha ligand maintained ERalpha selectivity in vivo during disease. ERalpha ligand treatment also induced favorable changes in autoantigen-specific cytokine production in the peripheral immune system [decreased TNFalpha, interferon-gamma, and interleukin-6, with increased interleukin-5] and decreased CNS white matter inflammation and demyelination. Interestingly, decreased neuronal staining [NeuN+ (neuronal-specific nuclear protein)/beta3-tubulin+/Nissl], accompanied by increased immunolabeling of microglial/monocyte (Mac 3+) cells surrounding these abnormal neurons, was observed in gray matter of spinal cords of EAE mice at the earliest stage of clinical disease, 1-2 d after the onset of clinical signs. Treatment with either estradiol or the ERalpha ligand significantly reduced this gray matter pathology. In conclusion, treatment with an ERalpha ligand is highly selective in vivo, mediating both anti-inflammatory and neuroprotective effects in EAE.

Figure 1.

Treatment with an ERα-selective ligand is highly selective in vivo during EAE. A, Treatment with the ERα ligand PPT induced expected biological responses on uterine weight (y-axis = uterine weight in grams). Uterine weight was increased with PPT given as daily subcutaneous injections at 10 mg/kg/d. The decrease in uterine weight with ovariectomy compared with sham surgery demonstrated the sensitivity of the technique in detecting differences in uterine weights associated with differences in estrogen levels. Treatment with a dose of estradiol known to induce a late pregnancy level of estradiol was used as a positive control for an increase in uterine weight, whereas treatment with vehicle alone served as the negative control. The uteri were removed at day 35–40 during EAE treatment with the indicated hormone (sham vehicle, n = 6; OVX vehicle, n = 12; OVX estradiol, n = 18; OVX PPT, n = 18). OVX PPT and OVX Estradiol, each compared with OVX Vehicle, ∗∗∗p < 0.0001. WT, Wild type. B, Uterine weights were examined in ovariectomized ERβ knock-out mice as in A. Uterine weights were increased with PPT treatment in ERβ knock-out mice (OVX vehicle, n = 9; OVX estradiol, n = 12; OVX PPT, n = 12). OVX PPT and OVX Estradiol, each compared with OVX Vehicle, ∗∗∗p < 0.0001. C, Uterine weights were examined in ovariectomized ERα knock-out mice as in A. Uterine weights were not increased with PPT treatment in ERα knock-out mice (OVX vehicle, n = 6; OVX estradiol, n = 4; OVX PPT, n = 6).

Figure 2.

Treatment with an ERα-selective ligand is sufficient to reduce the clinical severity of EAE. A, EAE clinical severity was decreased in ovariectomized, wild-type (WT) C57BL/6 female mice treated with PPT. Daily treatments of ovariectomized mice with injections of vehicle (negative control), estradiol (positive control), or PPT (10 mg/kg/d) began, and then 7 d later, active EAE was induced with MOG 35–55 peptide. Mean clinical scores were significantly reduced in both estradiol- and PPT-treated mice compared with vehicle treated (p < 0.0001, Friedman test). Data are representative from experiments repeated a total of five times. B, The decrease in the mean clinical scores of EAE by PPT treatment was not dependent on the presence of ERβ. Ovariectomized, ERβ knock-out C57BL/6 female mice were treated with either PPT, estradiol, or vehicle as in A. Mean clinical scores were significantly reduced in both estradiol- and PPT-treated mice compared with vehicle treated (p < 0.0001, Friedman test). Data are representative from experiments repeated a total of three times. C, PPT treatment in vivo during EAE remains highly selective for ERα. Ovariectomized female ERα knock-out C57BL/6 mice were treated as in A. In ERα knock-out mice, mean clinical scores were not significantly different in PPT-treated compared with vehicle-treated. PPT-treated wild-type mice served as a positive control for a PPT treatment effect within the experiment. Data are representative from experiments repeated a total of three times. Error bars indicate variability of clinical scores between mice within a given treatment group. n = 5 mice per each treatment group.

Figure 3.

Treatment with an ERα ligand reduced proinflammatory cytokine production by peripheral immune cells in ovariectomized, wild-type C57BL/6 female mice with EAE. EAE was induced as in Figure 2, and at day 40 after disease induction, mice were killed, and cytokine production by MOG 35–55 stimulated splenocytes was determined. TNFα, IFNγ, and IL6 levels were each significantly reduced with PPT treatment, whereas IL5 levels were increased with PPT treatment. Error bars indicate variability of cytokine values for splenocytes between individual mice within a given treatment group, with n = 5 mice for each treatment group. Data are representative of experiments repeated three times. ∗p < 0.05.

Figure 4.

Treatment with an ERα ligand reduced inflammation and demyelination in spinal cords of mice with EAE. A, Representative H&E-stained thoracic spinal cord sections (4× magnification) from normal (healthy control), as well as vehicle-, E2-, and PPT-treated EAE mice. Vehicle-treated EAE spinal cord shows multifocal to coalescing areas of inflammation in the leptomeninges and white matter, around blood vessels, and in the parenchyma of the white matter (areas of inflammation shown by arrows). No inflammation was observed in either E2- or PPT-treated EAE spinal cords. B, Luxol fast blue-stained region of dorsal column (square in A) of spinal cords (40× magnification). Intense demyelination in the white matter is seen in vehicle-treated EAE sections only. C, Anti-MBP-immunostained dorsal column demonstrated demyelination in the white matter of vehicle-treated EAE sections only. D, Increase in total number of infiltrating cells after induction of EAE was semiquantified by counting DAPI+ cells in the entire delineated white matter (including dorsal, lateral, and ventral funiculi) and presented as percentage of normal. Vehicle-treated EAE mice had a significant increase in white matter cell density compared with healthy normal control, whereas E2-treated and the ERα ligand (PPT)-treated groups did not. E, The extent of demyelination was compared by staining thoracic spinal cord sections with Luxol fast blue. Myelin density is presented as percentage of normal. Vehicle-treated mice EAE mice had a significant decrease in myelin density in the entire delineated white matter as compared with normal control, whereas E2-treated and PPT-treated groups did not. Number of mice, three per treatment group; number of T1–T5 sections per mouse, six; total number of sections per treatment group, 18. ∗∗Statistically significant compared with normals (p < 0.001), 1 × 4 ANOVAs. Data are representative of experiments repeated in their entirety on another set of EAE mice with each of the treatments. Error bars represent SE of variability of the indicated measure between sections of mice within each treatment group.

Figure 5.

Treatment with an ERα ligand preserved neuronal staining in gray matter of spinal cords of mice with EAE. A–D, Split images of thoracic spinal cord sections stained with NeuN (red) in i and Nissl in ii at 4× magnification, derived from normal healthy control mice (A), vehicle-treated EAE (B), E2-treated EAE mice (C), and ERα ligand (PPT)-treated EAE mice (D), each killed very early during EAE, 1–2 d after the onset of clinical signs. iii, Merged confocal scan at 40× of NeuN+ (red) and β3-tubulin+ (green) colabeled neurons from an area represented by dotted white square area in i. iv, A 40× magnification of Nissl-stained area in solid black square in ii. A decrease in NeuN+ immunostaining and Nissl staining was observed in the dorsal horn, intermediate zone, and ventral horn of vehicle-treated EAE mice (B) compared with normal controls (A). White arrows in Biii denote loss of NeuN+ staining. In contrast, EAE mice treated with either estradiol (C) or PPT (D) had preserved NeuN and Nissl staining. E, After quantification of neurons in the entire delineated gray matter of T1–T5 sections, NeuN+ immunolabeled neurons were significantly decreased, by nearly 25%, in vehicle-treated EAE mice compared with normal controls, but E2- and PPT-treated EAE mice were not statistically different from normal controls. Number of mice, three per treatment group; number of T1–T5 sections per mouse, six; total number of sections per treatment group, 18. ∗∗Statistically significant compared with normals (p < 0.001); 1 × 4 ANOVAs. Data are representative of experiments repeated in their entirety on another set of EAE mice with each of the treatments. Error bars represent SE of variability of the indicated measure between sections of mice within each treatment group.

Figure 6.

Treatment with an ERα ligand reduced CD45+ and Mac 3+ cells in white and gray matter of mice with EAE. A, Thoracic spinal cord sections from mice used in Figure 5 were coimmunostained with NF200 (green) and CD45 (red) at 10× magnification. Shown are partial images with white and gray matter from normal control, vehicle-treated EAE, E2-treated EAE, or ERα ligand (PPT)-treated EAE mice. LF, Lateral funiculus of white matter; GM, gray matter. The vehicle-treated EAE cords had large areas of CD45+ cells associated with reduced NF200 axonal staining in white matter compared with the normal control, whereas estradiol and ERα ligand-treated EAE mice had only occasional CD45 positivity, with intact NF200 axonal staining. B, Consecutive sections from the same mice were also coimmunostained with β3-tubulin (green) and Mac 3 (red), with the section of the ventral horn designated by the dotted line square area in A scanned at 40× magnification by confocal microscopy. Vehicle-treated EAE mice demonstrated markedly increased Mac 3 staining in ventral horn gray matter compared with normal control mice, with most of these Mac 3+ cells having the morphology of microglia (inset, 100× magnification). They were surrounding neuronal structures (white arrows). In contrast, E2- and ERα ligand (PPT)-treated EAE cord sections demonstrated less Mac 3 immunostaining compared with vehicle-treated EAE mice. C, After quantification, neurofilament-stained axon numbers in white matter were significantly lower in vehicle-treated EAE mice compared with normal mice, whereas E2- and PPT-treated EAE mice demonstrated no significant reduction in axon numbers. Axon number is presented as percentage of normal. ∗∗Statistically significant compared with normal (p < 0.001); 1 × 4 ANOVAs. D, Mac 3+ cells were analyzed by density measurements and represented as percentage of vehicle-treated groups. Compared with vehicle-treated EAE mice, both the E2-treated and PPT-treated had significantly lower Mac 3+ immunoreactivity in gray matter. Number of mice, three per treatment group; number of T1–T5 sections per mouse, four; total number of sections per treatment group, 12. ∗∗Statistically significant compared with normal (p < 0.001); 1 × 4 ANOVAs. Data are representative of experiments repeated in their entirety on another set of EAE mice with each of the treatments. Error bars represent SE of variability of the indicated measure between sections of mice within each treatment group.