XY sex chromosome complement, compared with XX, in the CNS confers greater neurodegeneration during experimental autoimmune encephalomyelitis.

Women are more susceptible to multiple sclerosis (MS) and have more robust immune responses than men. However, men with MS tend to demonstrate a more progressive disease course than women, suggesting a disconnect between the severity of an immune attack and the CNS response to a given immune attack. We have previously shown in an MS model, experimental autoimmune encephalomyelitis, that autoantigen-sensitized XX lymph node cells, compared with XY, are more encephalitogenic. These studies demonstrated an effect of sex chromosomes in the induction of immune responses, but did not address a potential role of sex chromosomes in the CNS response to immune-mediated injury. Here, we examined this possibility using XX versus XY bone marrow chimeras reconstituted with a common immune system of one sex chromosomal type. We found that experimental autoimmune encephalomyelitis mice with an XY sex chromosome complement in the CNS, compared with XX, demonstrated greater clinical disease severity with more neuropathology in the spinal cord, cerebellum, and cerebral cortex. A candidate gene on the X chromosome, toll-like receptor 7, was then examined. Toll-like receptor 7 expression in cortical neurons was higher in mice with XY compared with mice with XX CNS, consistent with the known neurodegenerative role for toll-like receptor 7 in neurons. These results suggest that sex chromosome effects on neurodegeneration in the CNS run counter to effects on immune responses, and may bear relevance to the clinical enigma of greater MS susceptibility in women but faster disability progression in men. This is a demonstration of a direct effect of sex chromosome complement on neurodegeneration in a neurological disease.

Neuroprotection mediated through estrogen receptor-alpha in astrocytes.

Estrogen has well-documented neuroprotective effects in a variety of clinical and experimental disorders of the CNS, including autoimmune inflammation, traumatic injury, stroke, and neurodegenerative diseases. The beneficial effects of estrogens in CNS disorders include mitigation of clinical symptoms, as well as attenuation of histopathological signs of neurodegeneration and inflammation. The cellular mechanisms that underlie these CNS effects of estrogens are uncertain, because a number of different cell types express estrogen receptors in the peripheral immune system and the CNS. Here, we investigated the potential roles of two endogenous CNS cell types in estrogen-mediated neuroprotection. We selectively deleted estrogen receptor-α (ERα) from either neurons or astrocytes using well-characterized Cre-loxP systems for conditional gene knockout in mice, and studied the effects of these conditional gene deletions on ERα ligand-mediated neuroprotective effects in a well-characterized model of adoptive experimental autoimmune encephalomyelitis (EAE). We found that the pronounced and significant neuroprotective effects of systemic treatment with ERα ligand on clinical function, CNS inflammation, and axonal loss during EAE were completely prevented by conditional deletion of ERα from astrocytes, whereas conditional deletion of ERα from neurons had no significant effect. These findings show that signaling through ERα in astrocytes, but not through ERα in neurons, is essential for the beneficial effects of ERα ligand in EAE. Our findings reveal a unique cellular mechanism for estrogen-mediated CNS neuroprotective effects by signaling through astrocytes, and have implications for understanding the pathophysiology of sex hormone effects in diverse CNS disorders.

Estrogen receptor-ß ligand treatment modulates dendritic cells in the target organ during autoimmune demyelinating disease.

Estrogens act upon nuclear estrogen receptors (ER) to ameliorate cell-mediated autoimmune disease. As most immunomodulatory effects of estrogens in EAE have been attributed to the function of ER-α, we previously demonstrated that ER-ß ligand treatment reduced disease severity without affecting peripheral cytokine production or levels of CNS inflammation, suggesting a direct neuroprotective effect; however, the effect of ER-ß treatment on the function of immune cells within the target organ remained unknown. Here, we used adoptive transfer studies to show that ER-ß ligand treatment was protective in the effector, but not the induction phase of EAE, as shown by decreased clinical disease severity with the preservation of axons and myelin in spinal cords. The analysis of the immune cell infiltrates in the CNS revealed that while ER-ß ligand treatment did not reduce overall levels of CNS inflammation, there was a decrease in the DC percentage, and these CNS DC had decreased TNF-α production. Finally, experiments using DC deficient in ER-ß revealed that the expression of ER-ß on DC was essential for protective effects of ER-ß ligand treatment in EAE. Our results demonstrate for the first time an effect of ER-ß ligand treatment in vivo on DC in the target organ of a prototypic cell-mediated autoimmune disease.

Oestrogen receptor beta ligand: a novel treatment to enhance endogenous functional remyelination.

Demyelinating diseases, such as multiple sclerosis, are characterized by inflammatory demyelination and neurodegeneration of the central nervous system. Therapeutic strategies that induce effective neuroprotection and enhance intrinsic repair mechanisms are central goals for future therapy of multiple sclerosis. Oestrogens and oestrogen receptor ligands are promising treatments to prevent multiple sclerosis-induced neurodegeneration. In the present study we investigated the capacity of oestrogen receptor ß ligand treatment to affect callosal axon demyelination and stimulate endogenous myelination in chronic experimental autoimmune encephalomyelitis using electrophysiology, electron microscopy, immunohistochemistry and tract-tracing methods. Oestrogen receptor ß ligand treatment of experimental autoimmune encephalomyelitis mice prevented both histopathological and functional abnormalities of callosal axons despite the presence of inflammation. Specifically, there were fewer demyelinated, damaged axons and more myelinated axons with intact nodes of Ranvier in oestrogen receptor ß ligand-treated mice. In addition, oestrogen receptor ß ligand treatment caused an increase in mature oligodendrocyte numbers, a significant increase in myelin sheath thickness and axon transport. Functional analysis of callosal axon conduction showed a significant improvement in compound action potential amplitudes, latency and in axon refractoriness. These findings show a direct neuroprotective effect of oestrogen receptor ß ligand treatment on oligodendrocyte differentiation, myelination and axon conduction during experimental autoimmune encephalomyelitis.

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

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.

Androgens regulate gene expression in avian skeletal muscles.

Circulating androgens in adult reproductively active male vertebrates influence a diversity of organ systems and thus are considered costly. Recently, we obtained evidence that androgen receptors (AR) are expressed in several skeletal muscles of three passeriform birds, the golden-collared manakin (Manacus vitellinus), zebra finch (Taenopygia guttata), and ochre-bellied flycatcher (Mionectes oleagieus). Because skeletal muscles that control wing movement make up the bulk of a bird's body mass, evidence for widespread effects of androgen action on these muscles would greatly expand the functional impact of androgens beyond their well-characterized effects on relatively discrete targets throughout the avian body. To investigate this issue, we use quantitative PCR (qPCR) to determine if androgens alter gene mRNA expression patterns in wing musculature of wild golden-collared manakins and captive zebra finches. In manakins, the androgen testosterone (T) up-regulated expression of parvalbumin (PV) and insulin-like growth factor I (IGF-I), two genes whose products enhance cellular Ca(2+) cycling and hypertrophy of skeletal muscle fibers. In T-treated zebra finches, the anti-androgen flutamide blunted PV and IGF-I expression. These results suggest that certain transcriptional effects of androgen action via AR are conserved in passerine skeletal muscle tissue. When we examined wing muscles of manakins, zebra finches and ochre-bellied flycatchers, we found that expression of PV and IGF-I varied across species and in a manner consistent with a function for AR-dependent gene regulation. Together, these findings imply that androgens have the potential to act on avian muscle in a way that may enhance the physicality required for successful reproduction.

An animal model of cortical and callosal pathology in multiple sclerosis.

The pathological and radiological hallmarks of multiple sclerosis (MS) include multiple demyelinated lesions disseminated throughout the white matter of the central nervous system (CNS). More recently, the cerebral cortex has been shown to be affected in MS, but the elucidation of events causing cortical demyelination has been hampered by the lack of animal models reflecting such human cortical pathology. In this report, we have described the presence of cortical gray matter and callosal white matter demyelinating lesions in the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Similar to the pathological lesions of MS patients, EAE lesions have been classified as type I-leukocortical, type II-intracortical and type III-subpial. All of these lesions had varying degrees of demyelination, inflammatory cells and reactive astrocytes. Similar to MS, cortical layers during EAE showed demyelination, microglia activation, synaptic protein alterations and apoptotic cells. In addition, the callosal white matter during EAE had many inflammatory demyelinating lesions and axon degeneration. Functional electrophysiological conduction analysis showed deficits in both myelinated and unmyelinated callosal axons during early and late EAE. The chronic EAE mouse model has features that mimic cortical and callosal pathology of MS, and can be potentially used to screen agents to prevent these features of disease.

Additive effects of combination treatment with anti-inflammatory and neuroprotective agents in experimental autoimmune encephalomyelitis.

We studied the effects of combination treatment with an anti-inflammatory agent, interferon (IFN)-beta, and a putative neuroprotective agent, an estrogen receptor (ER)-beta ligand, during EAE. Combination treatment significantly attenuated EAE disease severity, preserved axonal densities in spinal cord, and reduced CNS inflammation. Combining ERbeta treatment with IFNbeta reduced IL-17, while it abrogated IFNbeta-mediated increases in Th1 and Th2 cytokines from splenocytes. Additionally, combination treatment reduced VLA-4 expression on CD4+ T cells, while it abrogated IFNbeta-mediated decreases in MMP-9. Our data demonstrate that combination treatments can result in complex effects that could not have been predicted based on monotherapy data alone.

A role for sex chromosome complement in the female bias in autoimmune disease.

Most autoimmune diseases are more common in women than in men. This may be caused by differences in sex hormones, sex chromosomes, or both. In this study, we determined if there was a contribution of sex chromosomes to sex differences in susceptibility to two immunologically distinct disease models, experimental autoimmune encephalomyelitis (EAE) and pristane-induced lupus. Transgenic SJL mice were created to permit a comparison between XX and XY within a common gonadal type. Mice of the XX sex chromosome complement, as compared with XY, demonstrated greater susceptibility to both EAE and lupus. This is the first evidence that the XX sex chromosome complement, as compared with XY, confers greater susceptibility to autoimmune disease.

A female preponderance for chemically induced lupus in SJL/J mice.

Both spontaneous and chemically induced rodent models of autoimmune nephritis and autoantibody production have been explored to understand mechanisms involved in human systemic lupus erythematosus (SLE). While it has been known for decades that women are more susceptible than men to SLE, mechanisms underlying this female preponderance remain unclear. One chemically induced model involves injection of hydrocarbon oils such as pristane into otherwise normal mouse strains, which results in the development of autoantibodies and inflammation in organs such as kidney and liver. It is unknown whether lupus-like disease induced by chemicals would exhibit a sex bias in disease susceptibility. Here, we show that SJL/J female mice injected with pristane display greater mortality, kidney disease, serum anti-nuclear and anti-dsDNA antibodies than their male siblings. This is the first evidence that a female sex bias exists in a chemically induced lupus model.