Medium-dose estrogen ameliorates experimental autoimmune encephalomyelitis in ovariectomized mice.
Abstract
Medium-dose estrogen ameliorates experimental autoimmune encephalomyelitis in ovariectomized mice: Estrogen is a neuro-protective hormone in various central nervous system (CNS) disorders. The present study evaluated the role of estrogen during experimental autoimmune encephalomyelitis (EAE) at doses selected to mimic any suppressive potential from the hormone during pregnancy. Here, mice were ovariectomized and then 2 weeks later treated with MOG antigen to induce EAE. Concurrently, mice then received (subcutaneously) an implanted pellet to deliver varying estrogen amounts over a 21-day period. Clinical scores and other parameters were monitored daily for the 21 days. At the end of the period, brain/spinal cord histology was performed to measure lymphocyte infiltration; T-cell profiles were determined through ELISA, flow cytometry, and real-time PCR.
Transcription factor expression levels in the CNS were assessed using real-time PCR; T-cell differentiation was evaluated via flow cytometry. The results demonstrated that estrogen inhibited development of EAE. Histological studies revealed limited leukocyte infiltration into the CNS.
High and medium dose of estrogen increased TH2 and Treg cell production of interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-β, but concurrently resulted in a significant reduction in production of interferon (IFN)-γ, IL-17, and IL-6. Flow cytometry revealed there were also significant decreases in the percentages of TH1 and TH17 cells, as well as significant increase in percentages of Treg and TH2 cells in the spleen and lymph nodes. Real-time PCR results indicated that high- and medium-dose estrogen treatments reduced T-bet and ROR-γt factor expression, but enhanced Foxp3 and GATA3 expression. Collectively, these results demonstrated that a medium dose of estrogen – similar to a pregnancy level of estrogen – could potentially reduce the incidence and severity of autoimmune EAE and possibly other autoimmune pathologies.
Introduction; Medium-dose estrogen ameliorates experimental autoimmune encephalomyelitis in ovariectomized mice.
Multiple sclerosis (MS) is a chronic autoimmune disease, distinguished by inflammation and demyelination in the central nervous system (CNS) that mainly affects young adults (MarkovicPlese & McFarland 2001; Bodhankar et al. 2013). The etiology of this heterogeneous disease has been related to immunologic, environmental, and genetic factors, but its precise cause is still unknown (Milo & Kahana 2010). The pathogenesis of the disease is accompanied by activation and infiltration of mononuclear cells predominantly antigen-specific CD4þ and CD8þ T-cells and Bcells in the CNS (Mosayebi et al. 2010; Zhang et al. 2014).
Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model for the study of MS. This model causes inflammation and demyelination similar to the disease manifestation seen in humans (Zhu et al. 2011; Markoullis et al. 2012). EAE is a T-cell-mediated autoimmune disease of the CNS induced by immunizing animals with myelin antigens.
Disease development is mediated by interferon (IFN)-c and interleukin (IL)-17-producing T-cells while IL-10 is critical in negative regulation (Park et al. 2005). T-cells generated through this immunization are able to pass through the blood–brain barrier (BBB) and propagate an influx of monocytes into the CNS. Subsequent activation of resident microglia and perhaps CNS cells, such as astrocytes lead to the demyelination of axons and axonal loss (Miller & Karpus 2007).
Regulatory T (Treg) cells expressing transcription factor (TF) Foxp3 are a CD4þ subset with potent immune suppressive activities that are decisive for maintaining self tolerance and immune homeostasis (Fontenot et al. 2005; Zhou et al. 2009). Treg cells play an important protective role in EAE based on the finding that mice depleted of Treg cells exhibit increased susceptibility to EAE, whereas adoptive transfer of Treg cells reduced EAE incidence (Akirav et al. 2009; Bebo et al. 2009; Stephens et al. 2009).
Materials and methods; Animals
C57BL/6 mice (female, 8-week-old) were obtained from the Pasture Institute of Iran (Tehran, Iran). Mice were housed in animal facilities of the BuAli Research Institute maintained at 23 ± 2 C with a 50 ± 5% relative humidity and a 12 h light/dark cycle. All mice had ad libitum access to standard rodent chow and filtered tap water. All experiments were performed according to Mashhad University of Medical Science ethical guidelines.
Surgery; Medium-dose estrogen ameliorates experimental autoimmune encephalomyelitis in ovariectomized mice.
To perform the ovariectomy, 10-week-old mice were anesthetized with an intraperitoneal injection of ketamine (150 mg/kg) and xylazine (0.1 mg/kg). Anesthesia was confirmed by a reduced respiratory rate and no response to gentle pinching of footpad. A ventral incision was made through the skin of the flank and the ovaries and ovarian adipose tissue were then aseptically removed. Ovaries were isolated by ligation of the most proximal portion of the oviduct before removal. The animals were then returned to their cages and monitored for recover from surgery. A single subset of mice underwent a sham operation and then would serve as the EAE only control for these studies. Surgery was performed 2 weeks prior to EAE induction.