Pregnancy level of estrogen attenuates experimental autoimmune ...

Journal of Neuroimmunology 277 (2014) 85–95

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Pregnancy level of estrogen attenuates experimental autoimmune encephalomyelitis in both ovariectomized and pregnant C57BL/6 mice through expansion of Treg and Th2 cells Dariush Haghmorad a,e, Abbas Ali Amini b,e, Mohammad Bagher Mahmoudi c, Maryam Rastin a, Mahmoud Hosseini d, Mahmoud Mahmoudi a,⁎ a

Immunology Research Center, BuAli Research Institute, Department of Immunology and Allergy, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Inflammation and Inflammatory Diseases Research Center, Department of Immunology and Allergy, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran c Department of Genetics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran d Neurocognitive Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran e Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran b

a r t i c l e

i n f o

Article history: Received 16 August 2014 Received in revised form 7 October 2014 Accepted 9 October 2014 Keywords: Pregnancy Multiple sclerosis Estrogen Central nervous system

a b s t r a c t Pregnancy suppressive effect on autoimmune diseases including Multiple Sclerosis and Rheumatoid Arthritis may result from high levels of sex steroids such as estrogen and estriol. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of estrogen on MS alleviation. Female C57BL/6 mice were immunized with MOG35–55. Clinical scores and other relevant parameters were monitored daily. Brain and spinal cord histology was performed to measure lymphocyte infiltration and central nervous system demyelination. Th1/Th2/Th17 and Treg cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed by real-time PCR and T cell differentiation was explored through flow cytometry examination. Pregnancy and pregnancy level of estrogen alleviated clinical manifestations in EAE induced mice, reduced CNS demyelination and cell infiltration, suppressed spleen T cell proliferation, enhanced production of anti-inflammatory cytokines in splenocytes and increased the percentage of Th2 and Treg cells. Furthermore, the results of real-time PCR for transcription factors and related cytokines of Th1/Th2/Th17 and Treg cells in CNS showed reduced expression levels of Th1 and Th17 transcription factors, including T-bet and ROR-γt, and decreased Th1 and Th17 cytokines including IFN-γ, TNF-α, IL-17 and IL-23. These results are the first to indicate that pregnancy and pregnancy level of estrogen ameliorate the EAE condition by favoring Treg and Th2 differentiation through induced expression of Foxp3 and GATA3 in the CNS. Moreover, pregnancy and pregnancy level of estrogen decreased mRNA levels of T-bet and ROR-γt in the CNS. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis is a chronic inflammatory disease of the central nervous system with pathological manifestations which consist of demolition of myelin, axonal injury, and oligodendrocytes loss. The prevalence of multiple sclerosis in females is two to three times higher than males; and clinical signs usually occur in the reproductive years. Females typically indicate a relapsing–remitting form, while males appear to have a more rapidly progressive form (Shu et al., 2013; Rejdak et al., 2010; Klose et al., 2013; Romme Christensen et al., 2012; Zhang et al., 2014).

⁎ Corresponding author at: Immunology Research Center, BuAli Research Institute, Department of Immunology and Allergy, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Fax: +98 511 7112596. E-mail address: [email protected] (M. Mahmoudi).

http://dx.doi.org/10.1016/j.jneuroim.2014.10.004 0165-5728/© 2014 Elsevier B.V. All rights reserved.

MS shares immunologic, histopathologic and clinical similarities with its animal model, experimental autoimmune encephalomyelitis (EAE) (Markoullis et al., 2012). EAE has provided valuable information into immunopathogenic mechanisms of MS and is regarded as Th1 and Th17 mediated autoimmune disease wherein pro-inflammatory T cells, followed by macrophages, penetrate into the CNS and lead to demyelination and axonal death (Croxford et al., 2011; Herz et al., 2010; Papenfuss et al., 2004). During antigen presentation, dendritic cells release cytokines that direct naïve T cells to differentiate into one of the distinct developmental pathways, including generation of Th1, Th2, Th17 or Treg cells (Egwuagu and Larkin, 2013). Primarily, it was shown that IFN-γ producing Th1 cells were the predominant pathogenic cells in EAE and afterward, it was recognized that Th17 cells like Th1 cells have an important pathogenic role in the development of EAE (El-behi et al., 2010; Cua et al., 2003). IL-10 which mainly is produced by Th2 cells suppresses Th1/Th17 cells and prevents the production of pro-inflammatory

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D. Haghmorad et al. / Journal of Neuroimmunology 277 (2014) 85–95

cytokines including IFN-γ, TNF-α and IL-1. It seems that IL-10 is a potent anti-inflammatory cytokine that effectively modulates EAE (Moore et al., 2001; Asadullah et al., 2003). Treg cells are another important subpopulation of CD4+ T cells that have a pivotal role in preserving immune homeostasis and tolerance (Shevach, 2009; Bennett et al., 2001; Bebo et al., 2009). Substantial gender distinctions exist in the incidence of human autoimmune diseases. The prevalence of certain autoimmune diseases such as Systemic Lupus Erythematosus, Rheumatoid Arthritis, Graves's disease, and Multiple Sclerosis in females is notably different in comparison to males (Fairweather et al., 2008; Martocchia et al., 2011). Although the incidence of MS is higher in women, its relapse rates decline during late pregnancy, and treatment with pregnancy levels of estrogen reduces brain and spinal cord damages. Pregnancy is identified by physiological changes that could establish both neuroprotective and immunomodulatory influences and one such effect is a systemic shift from Th1-type cellular immunity toward Th2-type humoral immunity (Vukusic et al., 2004; Soldan et al., 2003). Previous studies have documented that treatment with different doses of estradiol could ameliorate EAE in both female and male mice as well as in numerous susceptible mouse strains to EAE (Elloso et al., 2005; Subramanian et al., 2003). The therapeutic effects of estrogen in EAE include protection against demyelination and also reduction in CNS inflammatory cells. Additionally, estrogen treatment modifies cytokine production, growth factors, adhesion molecules, chemokine and chemokine receptors in EAE induced mice (Offner, 2004; Matejuk et al., 2003). The impact of ovariectomy on EAE is argumentative. Some researchers found no significant outcome of ovariectomy on EAE progression and others reported a worsening of EAE development thereby recommending advantageous effect of endogenous estrogens (Voskuhl and Palaszynski, 2001). Female sex steroids appear to have decisive impacts on multiple sclerosis disease activity. Pregnancy contributes to remission of MS and decreased rates of flare, which increase markedly after parturition (Hughes, 2004). Multiple studies have reported a general alleviation in MS clinical disease activity during pregnancy documented from large-scale human studies in pregnant women with relapsing– remitting MS (McClain et al., 2007). Pregnancy also ensures protection of female animal models with EAE. Specifically, pregnant SJL and C57BL/ 6 mice showed disease amelioration compared to non-pregnant controls (Gatson et al., 2011; McClain et al., 2007). This study concentrates on the immunologic, histopathologic and clinical changes that occur during treatment with endogenous and exogenous estrogen in mice suffering from EAE and further looking into the role of estrogen in ameliorating mice with established EAE. Ovariectomized mice were used in order to avoid the potential challenge of several circulating sex hormones. The suppressive potential of pregnancy level estrogen could improve the medication plans for MS and other autoimmune diseases. 2. Material and methods 2.1. Animals Female C57BL/6 mice were purchased from Pasture Institute of Iran, and were used at 8 to 12 weeks of age. Mice were housed in animal facilities of the BuAli Research Institute and maintained on a 12-h light/ dark cycle. All animal procedures and care were carried out in accordance with ethical guidelines approved by Mashhad University of Medical Science.

performed through the skin of the flank of mice, and the ovaries and ovarian adipose tissue were removed. The most proximal portion of the oviduct ligated and then ovaries were removed. Surgery was carried out two weeks prior to EAE induction. The animals were returned in their cages to recover from surgery. Female ovariectomized mice were implanted with 15 mg/21-day release 17β-estradiol pellets (Innovative Research of America, Sarasota, FL, USA) and control animals were implanted with placebo pellets containing vehicle one day prior to EAE induction. Placebo pellets contain all the components of hormone pellets except the 17β-estradiol itself. For pregnancy experiments, 5 days prior to EAE induction eight- to ten-week old females were housed with ten- to twelve-week old males in a ratio of 3:1 for mating and were left in cages for 72 h. Pregnancy was confirmed by the existence of a vaginal plug and weight increase (raise ≥25% over baseline). The concentration of estradiol in pellet treated animals is expected to be 9000–10,000 pg/ml which is comparable to the level of estradiol (5000–10,000 pg/ml) in pregnant mice (Bebo et al., 2001; Ito et al., 2001). Mice were divided into four groups: 1. Ovariectomized placeboimplanted group (OVA) (n = 9), 2. Ovariectomized estradiolimplanted group (EST) (n = 10), 3. Virgin group (WT) (n = 10) and 4. Pregnant group (PRG) (n = 9 after verifying pregnancy). 2.3. Induction and clinical assessment of EAE All mice were immunized on day 0 by subcutaneous (s.c.) injection in the flanks with 250 μg MOG35 (MEVGWYRSPFSRVVHLYRNGK) (Chengdu Kaijie Biopharm Co. Ltd, China) emulsified in complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA) containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). Furthermore, 250 ng pertussis toxins (Sigma-Aldrich, St. Louis, MO, USA) were intraperitoneally injected on days 0 and 2 after immunization (Huehnchen et al., 2011). Clinical signs of EAE were monitored and the weight of the mice was measured daily until 21 days post immunization. Mice were scored for neurologic malfunction, in accordance with the following scale: 0, no clinical sign; 1, partial loss of tail tonicity; 2, complete loss of tail tonicity; 3, flabby tail and abnormal manner of walking; 4, hind leg paralysis; 5, hind leg palsy with hind body partial immobility; 6, hind and foreleg paralysis; and 7, moribund or death (Skundric et al., 2003; Sinha et al., 2008; Mosayebi et al., 2010; Toft-Hansen et al., 2011). Mice were scored daily and were assessed for incidence, onset day of disease, maximal score (at the peak day), mean score (at the last day) and cumulative disease index (total disease score over experiment duration). 2.4. Histopathology To estimate the rate of CNS inflammation and demyelination at the time of sacrifice (at day 21), mice were anesthetized with ketamine & xylazine and perfused by intracardiac injection of PBS containing 4% paraformaldehyde. Paraffin embedded 5 mm sections of the brain and the spinal cord were stained with hematoxylin and eosin (H&E) for the evaluation of inflammation and Luxol fast blue for demyelination. Sections are scrutinized by light microscopy in a blinded manner (Sloka et al., 2013; Berard et al., 2012). Inflammation was determined as follows; 0. No inflammation; 1. Small number of inflammatory cells; 2. Existence of perivascular infiltrates; and 3. Extending intensity of perivascular cuffing with expansion into contiguous tissue. Demyelination was scored as follows: 0. No demyelination thing; 1. Unique foci; 2. Few sections of demyelination; 3. Considerable sections of demyelination (Horstmann et al., 2013).

2.2. Surgery, estrogen treatment and pregnancy induction 2.5. Cell culture and BrdU proliferation assay Mice were anesthetized with ketamine (150 mg/kg) and xylazine (0.1 mg/kg). Anesthesia was verified by decreased respiratory rate and no response to mild nipping of the foot-pad. Ventral cutting was

Peripheral lymph nodes (inguinal and axillary) and spleens were isolated from C57BL/6 mice on day 21 post immunization. Cell suspensions


were prepared and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin in round-bottom 24-well plates (2 × 106 cells/well) and 96-well plates (4 × 105 cells/well). Cells were cultured in medium alone or with MOG35–55 (20 μg/ml). Cultures were incubated for 72 h at 37 °C and 5% CO2 and the last 24 h for 96-well plates in the presence of BrdU labeling solution (100 μl/ml). Proliferation was evaluated using a Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Applied Science, Indianapolis, IN, USA) in compliance with the manufacturer's instructions.

A

2.6. ELISA for cytokine detection Supernatants from 24-well plates were gathered after 72 h, and cytokine concentrations (IL-4, IL-6, IL-10, IL-17, TNF-α, TGF-β and IFNγ) were measured by ELISA according to the manufacturer's instructions (eBioscience, San Diego, CA, USA). Briefly, ELISA plates were coated with capture antibody diluted in coating buffer and incubated overnight at 4 °C. After washing wells were blocked with ELISA diluent for 1 h at room temperature. For cytokine detection, standards and supernatants were incubated at room temperature for 2 h, followed by 1 h of incubation

*

*

100 n= 9

EST

n=10

Incidence %

80

100 WT

Incidence %

OVA

87

60 40

80

n=10

PRG n= 9

60 40

20

20

0

0

Days Post Immunization


B 6

4

WT n=10

5

EST n=10

Clinicl Score

Clinicl Score

5

6

*** OVA n= 9

3 2

4 3 2

1

1

0

0 Days Post Immunization

**

PRG n= 9


C 21

Body Weight (g)

20 19

OVA n= 9 EST

*

n=10

18 17 16 15 Days Post Immunization

Fig. 1. Pregnancy level of estrogen inhibited the development of EAE in MOG-immunized C57BL/6 mice. Female ovariectomized C57BL/6 mice were implanted with 15 mg/21-day release 17β-estradiol pellets or were conceived prior to EAE induction with MOG35–55 as detailed under the Material and methods section. Mice were monitored for signs of EAE, and the results for all mice, were presented as (A) incidence of disease, (B) mean clinical score ± SEM, and (C) body weight for OVA and EST groups; for PRG and WT groups body weight was disregarded because of pregnancy and parturition. Results were expressed as mean ± SEM. *p b 0.05, **p b 0.01 and ***p b 0.001, compared with OVA and WT groups. Mice were divided into four groups: 1. ovariectomized placebo-implanted group (OVA), 2. ovariectomized estradiol-implanted group (EST), 3. virgin group (WT), and 4. pregnant group (PRG).

88


with biotinylated secondary antibody and then 30-minute incubation with Avidin-HRP. Plates were developed using tetramethylbenzidine (TMB). Reaction was stopped with stop solution and plates were measured at 450 nm using the microplate reader (Stat Fax 2100 Awareness, Phoenix, AZ, USA). Standard curves were calculated based on measurements of different concentrations of recombinant cytokines. 2.7. Flow cytometry Splenocytes were removed and suspended in complete RPMI 1640 with 10% FBS at a density of 5 × 106/ml. For intracellular cytokine staining, mononuclear cell suspensions were restimulated with PMA (phorbol 12-myristate 13-acetate) (50 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) and ionomycin (1 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) for 4 h in the presence of Brefeldin A, (10 mg/ml, BD Biosciences, San Jose, CA, USA). Cells were collected and washed in staining buffer. Following second wash step, cells were stained with a FITC anti-CD4 (GK1.5) and PE-Cy5 anti-CD8 (536.7) for 30 min at 4 °C. Cells were washed, fixed and permeabilized using fixation/permeabilization buffer (BD Biosciences, San Jose, CA, USA). At last, cells were stained for intracellular cytokines with PE-conjugated rat anti-mouse IL-4 (11B11), IFNγ (XMG1.2) and IL-17 (eBio17B7) antibodies. For intracellular staining of Foxp3, mononuclear cells washed in staining buffer and stained with a FITC anti-CD4 (GK1.5) and PE anti-CD25 (PC61.5) for 30 min at 4 °C. Cells were washed and incubated in fixation/permeabilization buffer (BD Biosciences, San Jose, CA, USA). Foxp3 staining was performed using the PE-Cy5 anti-Foxp3 (FJK-16s) antibody. All flow cytometric experiments were performed according to the manufacturer's instructions and using appropriate isotype controls. All antibodies were obtained from eBioscience (San Diego, CA, USA). Data were collected on a FACSCalibur (BD Biosciences, San Jose, CA, USA) and analyzed using CellQuest software. 2.8. Real-time PCR To determine cytokine expression and immune cell infiltration, both the spinal cord and brain were removed from diseased mice at day 21 after EAE induction. The tissues of each mouse were separately pulverized in phosphate buffer using a nylon mesh to detach cells. The suspension was centrifuged at 3000 g for 10 min and the resulting cell pellet was suspended in TriPure Isolation Reagent (Roche Applied Science, Indianapolis, IN, USA) for RNA extraction. cDNA synthesis was conducted by PrimeScript™ RT reagent Kit (Takara Bio Inc., Otsu, Shiga, Japan) in compliance with manufacturer's guidance. The mononuclear cells from spleen and lymph nodes were suspended in TriPure for RNA extraction and cDNA synthesis. Real-time PCR was accomplished by applying TaqMan PCR master mix (Takara Bio Inc., Otsu, Shiga, Japan) and specific primers for target genes including: IFN-γ (5′-CCAAGTTT GAGGTCAACA-3′ and 5′-CTGGCAGAATTATTCTTATTGG-3′), IL-4 (5′CTGGATTCATCGATAAGC-3′ and 5′-GATGCTCTTTAGGCTTTC-3′), IL-6 (5′-CCAACAGACCTGTCTATA-3′ and 5′-GCATCATCGTTGTTCATA-3′), IL10 (5′-CAGGTGAAGACTTTCTTTC-3′ and 5′-AACCCAAGTAACCCTTAA3′), IL-17 (5′-CCTCAGACTACCTCAACC-3′ and 5′-CCAGATCACAGAGG GATA-3′), IL-23 (5′-CGGGACATATGAATCTACTAA-3′ and 5′-TGTCCTTG AGTCCTTGTG-3′), TNF-α (5′-GGCTGCCCCGACTACGT-3′ and 5′-TTTCTC CTGGTATGAGATAGCAAATC-3′), TGF-β (5′-CGGACTACTATGCTAAAGA3′ and 5′-CTGTGTGAGATGTCTTTG-3′), Foxp3 (5′-CAGAGTTCTTCCAC AACA-3′ and 5′-CATGCGAGTAAACCAATG-3′), GATA3 (5′-CTGCGGAC TCTACCATAA-3′ and 5′-CATGCGAGTAAACCAATG-3′), T-bet (5′-TGTG GTCCAAGTTCAACC-3′ and 5′-CATCCTGTAATGGCTTGTG-3′), ROR-γt (5′-GGATGAGATTGCCCTCTA-3′ and 5′-CCTTGTCGATGAGTCTTG-3′). Reactions were conducted on the Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia) to detect the relative quantity of mRNA expression compared with the beta2 microglobulin (5′-CCTGTATGCTAT CCAGAA-3′ and 5′-GTAGCAGTTCAGTATGTTC-3′) reference gene.

2.9. Statistical analysis One-way, non-parametric analysis of variance (ANOVA) (Kruskal– Wallis test) followed by Dunn's multiple comparison tests was conducted for analysis of clinical signs between groups. Comparison of the effect of pregnancy and pregnancy level estrogen versus related control groups on the development of clinical signs was conducted via twoway repeated measures ANOVA. Mann–Whitney non-parametric unpaired t tests were used for two group comparisons. SPSS 19 was used to analyze the data. Data were presented as mean ± SEM. Statistical difference was accepted at the level of p b 0.05.

3. Results 3.1. Pregnancy and estrogen treatment alleviate the progression of clinical disease To determine the effects of pregnancy on established EAE, we induced pregnancy and immunized C57BL/6 mice with the immunodominant epitope of MOG 35–55 peptide. Moreover, mice were implanted with placebo or estradiol pellet one day prior to immunization with MOG. We observed suppression of disease throughout the later stages of pregnancy as compared to virgin group (WT); it seems that pregnant group (PRG) afforded a near full recovery from disease. Mice lacking ovary (ovariectomized placebo implanted group) developed more extensive EAE than WT and ovariectomized estradiol implanted group (EST). In contrast to the ovariectomized placebo-implanted group (OVA), the EST group developed EAE similar to PRG group and estradiol-implanted mice were almost completely protected against EAE through day 21 post-immunization similar to pregnant group. All mice (100%) in the WT and OVA groups developed severe EAE on approximately days 9 to 21. In contrast, less than 70% of mice in the PRG and EST groups showed mild signs of disease (Fig. 1A and Table 1). Besides, mice in the PRG and EST groups exhibited significant reduction in the severity of EAE. On day 17, when EAE clinical score reached the peak, the mean scores of PRG and EST groups were 1.3 and 1.7 respectively, in comparison with 4 and 4.5 for the WT and OVA groups respectively (Fig. 1B and Table 1). The EST group also markedly prevented the loss of body weight in EAE mice (Fig. 1C) and for PRG group, body weight was disregarded because of pregnancy and parturition. OVA group developed EAE more severe than WT group, but the difference was not significant. Both PRG and EST groups showed mild signs of disease and didn't show any significant difference in severity. These results suggested that estrogen in pregnancy level either endogenous or exogenous reduced the clinical severity in EAE mice.

Table 1 Clinical features of EAE in the administration of estrogen and pregnancy. Therapeutically treatment from onset Group Incidence Day of onset Maximal score Mean score (score at peak) (last day) EST OVA PRG WT

7/10 9/9 6/9 10/10

11.9 9.8 12.1 10.3

± ± ± ±

0.2⁎ 0.3 0.4⁎ 0.1

1.7 4.6 1.3 4

± ± ± ±

0.5⁎⁎⁎ 0.5 0.5⁎⁎ 0.4

0.85 4.1 0.7 3.4

± ± ± ±

Cumulative disease index (CDI)

0.3⁎⁎⁎ 11.6 ± 0.4⁎⁎⁎ 0.3 38.4 ± 1.95 0.2⁎⁎⁎ 9.8 ± 0.4⁎⁎⁎ 0.3 33.4 ± 1.3

EST: ovariectomized estradiol-implanted versus OVA: ovariectomized placebo-implanted. PRG: pregnant versus WT: Virgin. Data were expressed as mean ± SEM. ⁎ p b 0.05. ⁎⁎ p b 0.01. ⁎⁎⁎ p b 0.001.


3.2. High dose estrogen reduces CNS demyelination and limits CNS cell infiltration during established EAE Mice were sacrificed 21 days post immunization and the mean clinical score at the time of sacrifice was as follows: OVA (4.1 ± 0.3), EST (0.85 ± 0.1), WT (3.4 ± 0.5) and PRG (0.6 ± 0.2). To confirm the effect of estrogen on CNS pathology, we detached and examined the spinal cord of mice. To evaluate mononuclear cell infiltration and perivascular cuffing in MOG-immunized mice, spinal cord tissues were stained with hematoxylin and eosin. To evaluate demyelination during EAE, spinal cord sections were stained with Luxol fast blue (Fig. 2). Consistent with the clinical results, spinal cord sections from placebo-implanted mice demonstrated massive leukocyte infiltration with several foci of inflammation along with significant demyelination

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in the areas of the spinal cord similar to virgin mice, whereas both spinal cord sections from estradiol implanted mice and pregnant mice showed fewer infiltrating cells and areas of perivascular cuffing, and no obvious signs of inflammation and demyelination (Fig. 2). These results demonstrate conclusively that estrogen plays the major role in mediating pregnancy protection against EAE. 3.3. Absence of estrogen elicits a high pro-inflammatory environment in spleens and pregnancy level estrogen suppresses T-cell proliferation The cytokine milieu was evaluated in the spleens of all groups. MOG-specific responses generated in spleens were determined for cytokine expression by culturing mononuclear cells from mice and collecting supernatants after 72 h. Placebo-implanted mice produced

A

EST

OVA

PRG

WT T

B

ES ST

O OVA

PR RG

WT T

C

Pathological Score

3 2.5

ES ST VA OV PR RG W WT

* *

* *

2 1.5 1 0.5 0 Inflam matioon

Dem myelinnatioon

Fig. 2. Comparative histopathology of spinal cords demonstrated that pregnancy level of estrogen suppresses CNS inflammation and demyelination. Histopathological evaluation of spinal cords from implanted groups (placebo-implanted and E2-implanted mice) and pregnant groups (WT and Pregnant mice) was performed. Spinal cords from each group, collected on day 21 post-immunization, were fixed in paraformaldehyde and embedded in paraffin. Five micrometer sections from different regions of the spinal cord from each of the groups were stained (A) with H&E to enumerate infiltrating leukocytes and (B) with Luxol fast blue to assess demyelination. (C) CNS inflammatory foci and infiltrating inflammatory cells were quantified. Pathological scores including inflammation and demyelination were analyzed and shown with bar graph as mean scores of pathological inflammation or demyelination ± SEM. Data are representative of 3 independent experiments. *p b 0.05, **p b 0.01 and ***p b 0.001 compared with OVA and WT groups. Mice were divided into four groups: 1. ovariectomized placebo-implanted group (OVA), 2. ovariectomized estradiol-implanted group (EST), 3. virgin group (WT), and 4. pregnant group (PRG).

90


1

3.4. Estrogen enhanced anti-inflammatory cytokine production in splenocytes from EAE mice To determine if anti-inflammatory cytokines contributed to suppression of clinical disease observed during pregnancy or estrogen high dose treatment, splenocyte production of IL-4, IL-10 and TGF-β was examined. IL-4 was found to be produced in significant large amounts in both pregnant and estradiol implanted groups (Fig. 3B). IL-10 was significantly up-regulated in both pregnant and E2implanted mice as compared to control groups (OVA and WT). Results also showed higher level of TGF-β in pregnant and E2-implanted groups, which suggests that estrogen in high level, favors Treg differentiation (Fig. 3B) and could modulate T cell cytokine secretion. 3.5. Effects of pregnancy level estrogen on the polarization of CD4+ T cells in EAE mice To further investigate the possible role of pregnancy level estrogen in helper T cell polarization, we analyzed the CD4+ T cell subsets in

A

9000

***

Optical Density

higher levels of TNF-α, IL-6, IL-17 and IFN-γ as compared to their E2implanted counterparts (Fig. 3A). The splenocytes of pregnant mice produced the lowest levels of TNF-α, IL-6, IL-17 and IFN-γ. E2implanted mice produced almost equivalent levels of these cytokines as the pregnant mice. However, splenocytes of WT mice as well as placebo-implanted mice produced significantly higher levels of proinflammatory cytokines as compared to both E2-implanted and pregnant mice. These results suggest that treatment with estrogen could modulate cytokine secretion of T cells. Moreover, proliferation responses of splenic T cells were inhibited by pregnancy level of estrogen in E2-implanted and pregnant mice, but no inhibition was seen in WT and placebo-implanted mice (Fig. 4).

0.2

spleens from EAE mice. In accordance with flow cytometry results, there was a significant decrease of the percentage of IFNγ+ producing CD4+ (Th1 cell) and IL-17 producing CD4+ (Th17 cell), but a significant increase of the percentage of IL-4 producing CD4 + (Th2) and CD4 + CD25 + Foxp3 + (Treg cells) in splenocytes of pregnant and E2-implanted mice (Fig. 5). In addition, the expressions of Th1, Th2, Th17 and Treg specific transcription factors and related

OVA PRG WT

**

**

300 250

EST OVA

200 *

150

PRG

*

WT

100

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50 0

0 IL-17

IFN-γ 180

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IL-6

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4000

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160

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140 120

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**

PRG WT

Citokine (pg/ml)

Citokine (pg/ml)

WT

Fig. 4. Pregnancy level estrogen suppresses T-cell proliferation. Spleen cells were harvested on day 21 post-immunization and cultured in medium alone or with MOG (20 μg/ml) for 72 h on 96-well plates. Proliferation responses tested using a Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Applied Science, Indianapolis, USA). Proliferation assay was conducted in triplicate wells. Data presented as mean optical density ± SEM. *p b 0.05, **p b 0.01 and ***p b 0.001 compared with OVA and WT groups. Mice were divided into four groups: 1. ovariectomized placebo-implanted group (OVA), 2. ovariectomized estradiol-implanted group (EST), 3. virgin group (WT) and 4. pregnant group (PRG).

2000

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MOG induced

Citokine (pg/ml)

Citokine (pg/ml)

EST

3000

20

OVA

400

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***

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Proliferation assay by Brdu Method

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7000

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8000

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3000 EST

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OVA

2000

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1000 500 0 TGF-β

Fig. 3. Pregnancy level of estrogen suppressed MOG-specific pro-inflammatory cytokines and enhanced MOG-specific anti-inflammatory cytokine production in splenocytes and lymph nodes from EAE mice. Splenocytes and lymph nodes from immunized mice from all groups (38 mice) were isolated on day 21 post-immunization and restimulated with MOG35–55 (20 mg/ml) for 72 h. Culture supernatants were collected and indicated cytokine levels were measured by ELISA. Cytokine assays were conducted in duplicate wells. (A) Pro-inflammatory cytokines as IFN-γ, IL-17, TNF-α and IL-6 ± SEM and (B) anti-inflammatory cytokines as IL-4, Il-10 and TGF-β were measured from supernatants of cultures from splenocytes and lymph nodes. Results from lymph nodes were similar to splenocytes and data was not shown. Mice were divided into four groups: 1. ovariectomized placebo-implanted group (OVA), 2. ovariectomized estradiol-implanted group (EST), 3. virgin group (WT), and 4. pregnant group (PRG). Results were expressed as mean ± SEM. *p b 0.05, **p b 0.01 and ***p b 0.001 compared with OVA and WT groups.


91

A

Percent of Th1

35

**

30

**

EST OVA PRG WT

25 20 15 10 5 0

B Percent of Th17

6

EST OVA PRG WT

*

5

*

4 3 2 1 0

C

EST OVA

4

Percent of Th2

3.5

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PRG WT

*

3 2.5 2 1.5 1 0.5 0

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EST 14

Percent of Treg

12

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OVA PRG WT

10 8 6 4 2 0

Fig. 5. Flow cytometry profiles of spleen mononuclear cells. The percentage of Th1, Th2, Th17 and Treg cells in the CD4+ gate was analyzed by flow cytometry. Mononuclear cells were isolated from spleen at the time of sacrifice on day 21 post-immunization from mice induced EAE. (A) Mononuclear cells were stimulated with PMA and ionomycin in the presence of the Golgi inhibitor brefeldin A for 4 h, then stained and analyzed by flow cytometry for intracellular production of (A) IFN-γ, (B) IL-17, and (C) IL-4. Values in the bar graphs are the mean ± SEM. (D) For intracellular staining of Foxp3, mononuclear cells were washed and stained with anti-CD4 and anti-CD25 antibodies for 30 min at 4 °C. Cells were washed and incubated in fixation/permeabilization buffer. Foxp3 staining was performed using the anti-Foxp3 antibody. CD4+ T cells from spleen were gated and their CD25 and Foxp3 expressions were analyzed by flow cytometry. Values in the bar graphs are the mean ± SEM. *, p b 0.05; **, p b 0.01; and ***, p b 0.001 compared with OVA and WT groups. Mice were divided into four groups: 1. ovariectomized placebo-implanted group (OVA), 2. ovariectomized estradiol-implanted group (EST), 3. virgin group (WT) and 4. pregnant group (PRG).


cytokines in were examined by Real-Time PCR. The results are presented in Fig. 6 and indicate that estrogen significantly reduced T-bet, TNF-α, IFN-γ (Th1-related) and ROR-γt, IL-6, IL-17, IL-23

7

*

PRG

6

WT

5 4 3 2 1 0

**

35

OVA

WT 25 20 15 10 5

14 12 10 8 6 4 2

Relative expression

*

***

100

0.2

PRG WT

60 40 20

IL-17 EST

*

EST 2.5

OVA PRG

*

6

WT 5 4 3 2 1

OVA

* Relative expression (Relative to B2microglobulin mRNA)

Relative expression (Relative to B2microglobulin mRNA)

0.4

0

0

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PRG

2

WT

1.5

1

0.5

0

IL-23

ROR-γt *

*

4 3.5 3 2.5 2 1.5 1 0.5

EST OVA PRG WT

2

Relative expression

5

(Relative to B2microglobulin mRNA)

Relative expression

0.6

80

IL-6

4.5

0.8

OVA

***

0

7

1

EST OVA PRG WT

EST 120 (Relative to B2microglobulin mRNA)

Relative expression


16

EST OVA PRG WT

*

T-bet

IFN-γ

*

18

*

0

0

B


PRG

**

30

TNF-α

C

1.2

OVA

Relative expression

*

Relative expression

Relative expression


EST

EST

8


A

(Th17-related) expressions, but significantly enhanced GATA3, IL-4 (Th2-related) and Foxp3, IL-10, and TGF-β (Treg-related) expressions.


92

1.8 1.6

* *

1.4

EST OVA PRG WT

1.2 1 0.8 0.6 0.4 0.2 0

0

IL-4

GATA3

Fig. 6. mRNA expression of cytokines and transcription factors in CNS. On day 21 post-immunization, brains and spinal cords were collected and mRNA levels of cytokines and transcription factors were assessed by real time quantitative PCR as described in the Material and methods section. Assay was run in triplicate and relative expression of genes was determined compared to the housekeeping gene, β2 microglobulin. (A) Th1 related cytokines and transcription factors; TNF-α, IFN-γ and T-bet (B) Th17 related cytokines and transcription factors; IL-6, IL-17, IL-23 and ROR-γt (C) Th2 related cytokines and transcription factors; IL-4 and GATA3 (D) Treg related cytokines and transcription factors; IL-10, TGF-β and Foxp3. Results were expressed as mean ± SEM. *, p b 0.05; **, p b 0.01; and ***, p b 0.001 compared with OVA and WT groups. Mice were divided into four groups: 1. ovariectomized placebo-implanted group (OVA), 2. ovariectomized estradiol-implanted group (EST), 3. virgin group (WT) and 4. pregnant group (PRG).


3 2 1 0

*

7 6

*

5 4 3 2 1

EST OVA PRG WT

9

Relative expression

4

8


Relative expression


5

*

EST OVA PRG WT

Relative expression

*

6


D

93

8

* *

EST OVA PRG

7

WT

6 5 4 3 2 1 0

0

IL-10

TGF-

Foxp3

Fig. 6 (continued).

4. Discussion Pregnancy has been demonstrated to have suppressive effects on several autoimmune diseases, including MS and rheumatoid arthritis. Clinical improvements particularly occur during the third trimester of pregnancy in women with relapsing MS. Relapse rate increases following parturition but eventually returns to the pre-pregnancy level months after delivery (Confavreux et al., 1998; Vukusic et al., 2004). Our study concentrated upon reiterating the human clinical scenario in the mouse model to show the role of high level estrogen on EAE suppression during pregnancy. Pregnancy preservation involves sustained immune suppression and increased immune tolerance throughout the pregnancy period (Sabapatha et al., 2006). We found that there were significant changes in Th2 cytokines between pregnant and estrogen-implanted mice as compared to WT mice with preexisting EAE. The regulatory T cell product, IL-10, the most important immunosuppressive cytokine, has been shown to provide a powerful suppressive influence in mice immunized for EAE during pregnancy or in response to estrogen in mice with EAE (Bebo et al., 2001). In the present study, IL-10 was significantly upregulated in both pregnant and E2-implanted mice. We made use of two mice models to evaluate the therapeutic potential of high dose estrogen in MOG35–55 peptide immunized mice and observed their EAE disease severity. In pregnant mice, we investigated the natural estrogen's role in the improvement of EAE and in ovariectomized estradiol-implanted mice; we analyzed the exogenous estrogen effects in amelioration of disease progression without any interference of endogenous estrogen. We assessed the immune reaction defined by proliferation and cytokine expression of spleen and lymph node cells from MOG immunized mice. MOG specific T-cells, within the induction phase of EAE, move from draining lymph nodes to the spleen and subsequently into the CNS. Estrogen suppresses T-cell proliferation and IFN-γ production and reduces Ag-specific T cell proliferative responses, albeit not by direct interaction with T cells, and the mechanisms of this interplay remain unclear and controversial (Bodhankar et al., 2011). In addition, IL-10 could decrease inflammation by reducing MHC class II expression by APCs (Einstein et al., 2007). Lack of estrogen in the ovariectomized placebo-implanted mice led to a much more severe disease as determined by other EAE-related studies. Unlike estradiol-implanted mice, placebo-implanted mice demonstrated EAE disease scores and CNS damage which was similar to WT mice. On the other hand, while the absence of estrogen led to an EAE disease outcome more severe than WT mice, the E2-implanted mice were completely protected from developing EAE similar to the pregnant mice, with few infiltrating immune cells and low demyelination in the CNS. EAE is known to be predominantly a T cell-mediated autoimmune disease with IFN-γ and IL-17-producing T cell subsets responsible for

promoting EAE (Park et al., 2005). Pathogenic T cells are known to be induced in the periphery (spleens and draining lymph nodes) after which they migrate to the CNS to initiate an inflammatory process responsible for clinical signs of EAE. Studies have demonstrated that stimulated T cells from ovariectomized mice with active disease produced copious amounts of IL-17 and IFN-γ as compared to WT mice, suggesting that T cells with deficient estrogen signaling may be preferentially polarized toward Th1 and Th17 differentiation. We demonstrated that treatment with high level of estrogen is a powerful regulator of cytokines. It decreases the production of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6, and IL-17 and increases the production of anti-inflammatory cytokines, such as IL-4, IL-10 and TGF-β. Hence, ex vivo recall responses by means of cytokine production by splenocytes of the placebo-implanted and E2-implanted ovariectomized mice on day 25 were analyzed. Our results verified that, in the absence of estrogen, significantly higher MOG specific Th1/Th17 responses were generated in the periphery by the placebo-implanted mice. However, absences of estrogen led to a pro-inflammatory milieu more severe than WT mice. The cytokine profile in the EAE-protected and E2implanted mice was similar to that in the pregnant mice, demonstrating significantly lower expression of the pro-inflammatory cytokines TNFα, IFN-γ and IL17. In this study, we showed the effects of high dose estrogen (natural and artificial) on EAE model and explored its mechanism of action. Our results indicate that the severity and the development of EAE could be inhibited by both natural and artificial estrogen treatments from the day of immunization. Estrogen treatment markedly reduced the inflammation and demyelination in the active EAE model, and simultaneously reduced Th1, Th17 and up-regulated Treg cell percentage accumulation in the peripheral immune system. It is well established that Treg cells play an essential role in regulating autoimmune responses. Although TGF-β is commonly regarded as an immune suppressive mediator, it has bidirectional role. It is commonly accepted that TGF-β alone induces Treg cells, and maintains Treg cell survival; however, TGF-β in cooperation with IL-6 could persuade Th17 differentiation. Our results showed a higher level of TGF-β but lower level of IL-6 in pregnant and E2-implanted groups, which suggests that high level of estrogen favors Treg differentiation, but constrains Th17 development. In EAE model, Treg cells are reported to prevent inflammation through various pathways. Several studies have shown that Treg cell depletion prior to EAE induction enhances the severity of the disease and the production of IL-6, IL-17 and IFN-γ. This exhibits that Treg cells restrain the expansion of auto reactive effectors (O'Connor and Anderton, 2008). In the recovery phase of EAE, Treg cells are the main source of IL-10 in the CNS, in which its production is critical for complete recovery (McGeachy et al., 2005). Enhancing Treg cells and regulating the balance of Treg/Teff cells were used as therapeutic methods for autoimmune diseases (Shin et al., 2011).

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It is well known that Treg cells play an essential role in regulating autoimmune responses (Ephrem et al., 2008). In harmony with these findings, our results indicate that the protective influence of estrogen may also come from an increase in Treg numbers. While the molecular mechanisms by which estrogen shifts the balance between Th1/Th2 and Th17/Treg cells are currently unknown, for the first time, we demonstrated that the estrogen up regulates the expression level of transcription factors including GATA3 and Foxp3 in the CNS and derivate T cell to Th2 and Treg. The suppressive function of Treg cells is directed by Foxp3 and its high level expression is associated with suppressive function in both human and mouse CD4+ T cells (Liston and Gray, 2014). The prominent regulator of Th2 cells is GATA3 and is required for Th2 cell differentiation, stability, and cytokine production (Chen et al., 2014; Fernando et al., 2014). In conclusion, the current study has revealed that high dose estrogen treatment ameliorated the EAE severity by favoring Treg and Th2 cell expansion and functions, indicating the immunosuppressive mechanisms of estrogen. Our novel finding is that pregnancy and estrogen treated mice showed an increased expression of transcription factors GATA3 and Foxp3 versus decreased expression of transcription factors T-bet and ROR γt in the CNS. Indeed, this may explain the molecular mechanisms by which estrogen shifts the balance between Th1/Th2 and Th17/Treg cells. We have demonstrated that high dose estrogen treatment reduces the incidence and severity of clinical disease similar to pregnancy by favoring Treg and Th2 cell expansion. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment The authors would like to thank the authorities in the research council of Mashhad University of Medical Sciences (MUMS) for their financial support (Grant number 88735). References Asadullah, K., Sterry, W., Volk, H.D., 2003. Interleukin-10 therapy—review of a new approach. Pharmacol. Rev. 55, 241–269. Bebo Jr., B.F., Fyfe-Johnson, A., Adlard, K., Beam, A.G., Vandenbark, A.A., Offner, H., 2001. Low-dose estrogen therapy ameliorates experimental autoimmune encephalomyelitis in two different inbred mouse strains. J. Immunol. 166, 2080–2089. Bebo Jr., B.F., Dehghani, B., Foster, S., Kurniawan, A., Lopez, F.J., Sherman, L.S., 2009. Treatment with selective estrogen receptor modulators regulates myelin specific T-cells and suppresses experimental autoimmune encephalomyelitis. Glia 57, 777–790. Bennett, C.L., Christie, J., Ramsdell, F., Brunkow, M.E., Ferguson, P.J., Whitesell, L., Kelly, T.E., Saulsbury, F.T., Chance, P.F., Ochs, H.D., 2001. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27, 20–21. Berard, J.L., Zarruk, J.G., Arbour, N., Prat, A., Yong, V.W., Jacques, F.H., Akira, S., David, S., 2012. Lipocalin 2 is a novel immune mediator of experimental autoimmune encephalomyelitis pathogenesis and is modulated in multiple sclerosis. Glia 60, 1145–1159. Bodhankar, S., Wang, C., Vandenbark, A.A., Offner, H., 2011. Estrogen-induced protection against experimental autoimmune encephalomyelitis is abrogated in the absence of B cells. Eur. J. Immunol. 41, 1165–1175. Chen, X., Shen, Y., Liang, Q., Flavell, R., Hong, Z., Yin, Z., Wang, M., 2014. Effect of Bavachinin and its derivatives on T cell differentiation. Int. Immunopharmacol. 19, 399–404. Confavreux, C., Hutchinson, M., Hours, M.M., Cortinovis-Tourniaire, P., Moreau, T., 1998. Rate of pregnancy-related relapse in multiple sclerosis. Pregnancy in multiple sclerosis group. N. Engl. J. Med. 339, 285–291. Croxford, A.L., Kurschus, F.C., Waisman, A., 2011. Mouse models for multiple sclerosis: historical facts and future implications. Biochim. Biophys. Acta 1812, 177–183. Cua, D.J., Sherlock, J., Chen, Y., Murphy, C.A., Joyce, B., Seymour, B., Lucian, L., To, W., Kwan, S., Churakova, T., Zurawski, S., Wiekowski, M., Lira, S.A., Gorman, D., Kastelein, R.A., Sedgwick, J.D., 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744–748. Egwuagu, C.E., Larkin III, J., 2013. Therapeutic targeting of STAT pathways in CNS autoimmune diseases. Jakstat 2, e24134. Einstein, O., Fainstein, N., Vaknin, I., Mizrachi-Kol, R., Reihartz, E., Grigoriadis, N., Lavon, I., Baniyash, M., Lassmann, H., Ben-Hur, T., 2007. Neural precursors

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