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A natural flavonoid glucoside icariin inhibits Th1 and Th17 cell differentiation and ameliorates experimental autoimmune encephalomyelitis
International Immunopharmacology 24 (2015) 224–231
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A natural flavonoid glucoside icariin inhibits Th1 and Th17 cell differentiation and ameliorates experimental autoimmune encephalomyelitis Ruile Shen a,b,⁎, Wenjing Deng a, Chun Li a, Guangwei Zeng b a b
Department of Neurology, The First Affiliated Hospital of Zhengzhou University, China Department of Neurology, The First Affiliated Hospital of Henan University of Science and Technology, China
a r t i c l e
i n f o
Article history: Received 2 September 2014 Received in revised form 6 December 2014 Accepted 9 December 2014 Available online 17 December 2014 Keywords: Icariin Th1 cells Th17 cells EAE
a b s t r a c t Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Icariin, a natural flavonoid glucoside isolated from plants in the Epimedium family, has been proved to have various pharmacological activities. However, the effect of icariin on experimental autoimmune encephalomyelitis (EAE) has never been investigated. In our current study, we found that icariin treatment leads to alleviated inflammatory infiltration and reduced blood–brain barrier leakage (BBB) of the paracellular tracer (FITC-dextran) in EAE. Mice that received icariin-treated T cells also displayed lower EAE scores and better clinical recovery from EAE. Icariin administration suppresses the frequencies of Th1 and Th17 cells in the splenocytes and lymph node cells. Icariin-treated mice also show lower frequency of Th17 cells in CNS mononuclear cells. The effect of icariin on Th1 and Th17 cell differentiation may be mediated via modulation of dendritic cells (DCs). Furthermore, icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. In conclusion, icariin ameliorates EAE and this was associated with suppressed Th1 and Th17 cell differentiation. © 2014 Published by Elsevier B.V.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability [1]. CD4+ T cell mediated autoimmunity was suggested as one of the most important aspects of the pathogenesis [2]. Effector CD4+ T cells of Th1 and Th17 subsets are found in MS lesion and can mediate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression of Th1 and Th17 cytokines, IFN-γ and IL-17 is detected in MS lesions [3]. The role of IFN-γ and IL-17 in EAE has been extensively researched. EAE can be induced by the adoptive transfer of CNS antigen reactive Th1 cells [4–7] and Th17 cells [8–10]. Neutralization of IL-17 by treatment with anti-IL-17 antibodies has been demonstrated to reduce motor symptoms of EAE [11]. MiR-20b, as a small RNA, has been identified to suppress Th17 differentiation and the pathogenesis of EAE by targeting retinoid-related orphan receptor γt (RORγt) [12]. Other studies also demonstrated that targeting RORγt was a novel strategy for Th17 associated autoimmune diseases [13–17].
Icariin, a natural flavonoid glucoside isolated from plants in the Epimedium family, has been proved to have various pharmacological activities. Recent studies have indicated that icariin displayed positive effects on suppressing cardiac inflammation [18]. Icariin has also been demonstrated to suppress cartilage and bone degradation in a mouse model of collagen-induced arthritis [19]. However, the effect of icariin on EAE has never been investigated. In this study, we demonstrated a crucial role for icariin in suppressing Th1 and Th17 cell differentiation and ameliorating EAE. Icariin treatment leads to alleviated inflammatory infiltration and reduced BBB of the paracellular tracer (FITC-dextran) in EAE. Mice that received icariin-treated T cells also displayed lower EAE scores and better clinical recovery from EAE. Icariin administration suppresses the frequencies of both Th1 and Th17 cells in the splenocytes and lymph node cells. Icariintreated mice also show lower frequency of Th17 cells in CNS mononuclear cells. Furthermore, icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. 2. Materials and methods 2.1. Animals
⁎ Corresponding author at: Jinghua Road 24#, Jianxi District, Luoyang 471003, China. E-mail address: [email protected] (R. Shen).
http://dx.doi.org/10.1016/j.intimp.2014.12.015 1567-5769/© 2014 Published by Elsevier B.V.
Male C57BL/6 mice, all ages 6–8 weeks, were purchased from Center of Henan University of Science Technology. Mice were maintained in a
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specific pathogen-free condition. All experimental procedures were examined and approved by the Institutional Animal Care and Use Committee at Henan University of Science Technology. 2.2. Preparation of icariin Icariin (2-(4′-methoxylphenyl)-3-rhamnosido-5-hydroxyl-7glucosido-8-(3′-methyl-2-butylenyl)-4-chromanone, standard substance, HPLC N98%) was purchased from the Institute for Reference Standards and Standardization of National Institute for Food and Drug control (Beijing, China). Icariin was dissolved at a concentration of 30 mM in 100% DMSO as a stock solution, stored at − 20 °C, and diluted with a medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study (all the control groups are composed of 0.1% DMSO). 2.3. Induction of EAE Induction of EAE was conducted as described previously [20]. EAE was induced in C57BL/6 mice by immunization with 250 μg of MOG35–55 (myelin oligodendrocyte glycoprotein, MOG). All peptides were dissolved in complete Freund's adjuvant (CFA) containing 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra. At day 1 and 48 h after immunization, C57BL/6 mice were injected with 500 ng of pertussis toxin in PBS, intraperitoneally. Clinical assessment of EAE was performed after disease induction by the following criteria: 0, no disease; 1, tail paralysis; 2, hindlimb weakness or partial paralysis; 3, complete hindlimb paralysis; 4, forelimb and hindlimb paralysis; and 5, death. Icariin (25 mg/kg) was dissolved in 0.5% CMC solution and orally administered daily from day 5 to day 15 after immunization. For passive EAE experiments, donor mice were immunized with MOG/CFA in the same fashion as when inducing EAE, but no pertussis toxin was administered. Spleens and draining lymph nodes were collected 10 days later, single-cell suspensions were prepared, and RBCs were lysed. Cells (6 × 106 cells/ml) were cultured in RPMI1640 medium with 40 μg/ml of MOG35–55 peptide and 10 ng/ml of recombinant mouse IL-12 (R&D Systems). After 3 days of culture, cells were harvested and T cells were isolated by negative selection using Dynabeads (Invitrogen). Recipient mice were irradiated sublethally (500 cGy) and received 5 × 106 cells intravenously [21]. 2.4. Histopathology Mice were humanely euthanatized and spinal cord immersed in 4% paraformaldehyde for 48 h at 4 °C and paraffin embedded. Six micrometer thick (6 mm) longitudinal sections of the spinal cord were cut to include the majority of the length of the spinal cord (from cervical to lumbar regions), containing both gray and white matters. Prior to staining, sections were deparaffinized in xylene (2 × 5 min) and hydrated in graded ethanol (2 × 5 min in 100%, 5 min in 85%, 5 min 70%) to distilled water and finally rinsed in PBS. Spinal cord sections were stained with Hematoxylin/Eosin (Sigma, MO, USA) and with Luxol fast blue (American Mastertech, CA, USA) following the manufacturer's recommendations to assess cell infiltration and demyelination, respectively [22]. Four to five different sections per mouse were analyzed under light microscopy. At least 10 randomly distributed 40× fields within the white matter of the spinal cord were captured for each section. To determine the demyelination, the white matter was outlined automatically using the wand tool. The areas covered by the Luxol fast blue stain were quantified using Image-Pro Plus software (Media Cybernetics, MD, USA). The demyelination was determined as (Total white matter area − Luxol fast blue area) / Total white matter area × 100%. 2.5. In vivo BBB permeability assays On day 25 after induction of EAE, BBB permeability was determined by a published method [23]. Briefly, 70-kDa FITC-dextran (250 mg/kg)
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was injected intravenously, and 30 min later, the mice were transcardially perfused with 0.9% saline (400 ml/kg) and then with 4% paraformaldehyde (PFA, 800 ml/kg). Lumbar enlargement segments (L4–L5) were cross-sectioned at 20 μm thickness and mounted with DAPI-Fluoromount G. To determine the extravascular intensity of the FITC-dextran, images were obtained with fluorescence microscopy at ×40. The images were quantified with ImageJ software and normalized to fluorescence value in the liver and to tissue area. 2.6. Proliferative responses of T cell and cytokine analysis Spleens or draining lymph nodes were harvested and pooled from EAE mice, and single-cell suspensions were prepared. Cells were cultured at 5 × 107 cells/well in 24-well U-bottom plates with 10 mg/ml MOG35–55 peptide in complete RPMI 1640 medium. For ELISA analysis, supernatants were harvested at 72 h of culture. The concentrations of indicated cytokines were measured by quantitative capture ELISA, according to the guidelines of the manufacturers. For the detection of proliferative response, splenocytes or lymph node (LN) cells were seeded in 96-well plate at 5 × 103 cells/well with 10 mg/ml MOG35–55 peptide and then cultured for 72 h. 10 μl of CCK-8 (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan) solution was added to each well per plate, and incubated the plate at 37 °C for the final 4 h. The absorbance at 450 nm was assayed for proliferation. 2.7. Preparation and evaluation of CNS cells Brains of mice, which were perfuse with cold PBS, were dissected and incubated in 2.5 mg/ml collagenase D for 30 min at 37 °C. Singlecell suspensions were prepared. Cells were washed in RPMI 1640 medium, and mononuclear cells were isolated using a discontinuous Percoll gradient [24]. 2.8. Real time-PCR Total RNA was extracted from cultured cells or tissues using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. mRNA levels of target genes were quantified using SYBR Green Master Mix (Takara Biotechnology, Dalian, China) with ABI PRISM 7900 Sequence Detector system (Applied Biosystems, Foster City, CA). Each reaction was performed in duplicate, and changes in relative gene expression normalized to 18sRNA levels were determined using the relative threshold cycle method. 2.9. Flow cytometry analysis Isolated mononuclear cells from spleen, LN and CNS were cultured in 24-well plates in RPMI1640 medium supplemented with 10% FBS, 200 ng/ml phorbol myristate acetate (PMA, Sigma, St. Louis, MO), and 400 ng/ml ionomycin and brefeldin A (Sigma) for 4 h. The cells were harvested and stained with FITC-anti-human CD4 at 4 °C for 30 min. After washing with PBS, the cells were fixed, permeabilized, and stained with APC-anti-IL-17 or APC-anti-IFN-γ (eBioscience, San Diego, CA) at 4 °C for 30 min. The frequencies of Th17 and Th1 cells were analyzed using a FACS cytometer equipped with CellQuest software (BD Pharmingen). 2.10. MTT cell proliferation assay Purified T cells were cultured in 96-well plates at a density of 3 × 105 cells/well in RPMI 1640 medium and stimulated with 5 μg/mol Con A in the presence of various concentrations of icariin for 72 h at 37 °C. 20 μl of MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) (4 mg/ml in RPMI 1640) was added per well 4 h before the end of incubation. MTT formazan production was dissolved by DMSO
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and the optical density at 570 nm (OD570) was measured by a microplate reader. 2.11. In vitro Th1 and Th17 cell differentiation Spleens were harvested from naive C57BL/6 mice, and single-cell suspensions were prepared. CD4+ T cells were negatively selected using the EasySep Mouse CD4+ T Cell Enrichment Kit (STEMCELL Technologies). For T-cell differentiation into Th1, or Th17, cells were labeled with anti-CD4, anti-CD44, anti-CD62L, and anti-CD25 for cell sorting. CD4+CD25−CD44loCD62L+ naive T cells were isolated by flow cytometry. Cells were stimulated with 5 μg/ml Con A under Th1 (10 ng/ml mouse IL-12, 10 μg/ml anti-mouse IL-4, 40 U/ml mouse IL-2), or Th17 conditions (2 ng/ml TGF-β, 20 ng/ml mouse IL-6, 10 μg/ml anti-mouse IL-4, 10 μg/ml anti-mouse IFN-γ, 10 ng/ml mouse IL-23) for 5 days in the presence of icariin at the indicated concentration. The frequencies of Th17 and Th1 cells were analyzed using a FACS cytometer equipped with CellQuest software (BD Pharmingen).
serum concentration of icariin is 0.78 ± 0.32 μmol/ml) decreased the EAE scores and enhanced clinical recovery from EAE (Fig. 1A). Histological analysis of spinal cord sections showed that mice treated with control PBS developed prominent inflammatory infiltration and demyelination, whereas icariin treatment showed much fewer infiltration and significantly alleviated demyelination (Fig. 1B). Next, we investigated whether icariin treatment is in part associated with a decrease in vascular leakage in the region of inflammation. We observed that the icariin treatment showed similar levels of leakage of 70-kDa FITCdextran in control mice with no EAE. After induction of EAE, the soluble marker was found to be diffused through the spinal cord parenchyma and was of high abundance in control group. In contrast to control group, the icariin treatment showed a 50% reduction in BBB leakage of the paracellular tracer (FITC-dextran) (Fig. 1C). We then transferred T cells from MOG-immunized control or icariin-treated mice into wildtype recipients. Mice that received icariin-treated T cells displayed lower EAE scores and better clinical recovery from EAE (Fig. 1D). 3.2. Icariin inhibits Th1 and Th17 cell differentiation in EAE
2.12. Statistical analysis All data were presented as means ± SEM. ANOVA analysis was used for statistical analysis with P b 0.05 being considered statistically significant. Data were analyzed using Prism software (GraphPad Software, Inc.). 3. Results 3.1. Icariin ameliorates EAE development In the present study, we used a mouse model of EAE to evaluate the therapeutic effect of icariin. Results showed that icariin treatment (with
We examined the presence of IL-17 (Th17) or interferon-γ (IFN-γ) (Th1)-producing CD4+ T cells during EAE in lymph nodes, spleen and the CNS. Lymph nodes, spleen and CNS of mice were harvested on day 25 after immunization with MOG35–55. Intracellular cytokine staining showed that the frequencies of both Th1 and Th17 cells in the splenocytes and lymph node cells were lower in icariin-treated mice than that in controls (Fig. 2A–B). Icariin-treated mice had lower frequency of Th17 cells in CNS mononuclear cells compared to respective control. But frequency of Th1 cells was not changed in the CNS between the two groups (Fig. 2C). CD4+ T cells in control-treated mice underwent cell divisions in vitro with MOG35–55, whereas CD4+ T cells in icariin-treated mice
Fig. 1. Icariin ameliorates EAE development. A, clinical scores for mice treated with icariin after the onset of EAE; B, histology of paraffin sections of spinal cords isolated from oligonucleotide-infected mice on day 25 after immunization. Spinal cord sections were stained with H&E and Luxol fast blue to assess inflammation and demyelination; C, at day 25 after induction, mice were injected intravenously with 70-kDa FITC-dextran (250 mg/kg), and lumbar spinal cords and livers were obtained after perfusion with fixative. Images of cross sections of spinal cords at the L4–L5 levels were analyzed to determine extravascular fluorescence intensity. Quantification of extravascular intensity of 70-kDa FITC-dextran. For each mouse, the fluorescence intensity of spinal cord sections was normalized to that of liver sections from the same animal; and D, MOG-primed T cells from control or icariin-treated mice were transferred into sublethally irradiated mice. The mice were scored for clinical signs of EAE and the mean clinical score was calculated by averaging the scores of all mice. Arrows indicate inflammatory cell infiltrate. *P b 0.05. N = 6.
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Fig. 2. In vivo generation of Th17 cells and Th1 cells in EAE inhibited by icariin. A, intracellular staining of IL-17 and IFN-γ in spleen cells; B, intracellular staining of IL-17 and IFN-γ in LN cells; and C, intracellular staining of IL-17 and IFN-γ in CNS infiltrating mononuclear cells. Isolated cells were fixed, permeabilized, and stained with APC-anti-IFN-γ and APC-anti-IL-17. The frequencies of Th17 and Th1 cells were analyzed using a FACS cytometer equipped with CellQuest software (BD Pharmingen). *P b 0.05. N = 6.
had a reduced proliferation (Fig. 3A). In parallel, CCK8 assays via splenocytes produced similar differences (Fig. 3B). Splenocytes and LN cells of icariin-treated mice showed decreased production of both of IL-17A and IFN-γ compared to their controls (Fig. 3C). Genes controlling the expression of IL-17A and IFN-γ were also significantly downregulated in icariin-treated mice (Fig. 3D). Myeloid-derived suppressive cells (MDSCs) are a heterogeneous group of myeloid cells comprised of hematopoietic progenitor cells and precursors of macrophages, granulocytes, and dendritic cells (DCs) [25]. Recently, icariin has been reported to exert antiinflammatory effects via modulation of MDSCs [26] and DCs have been demonstrated as a therapeutic target in MS and EAE [27]. Therefore, we tested whether icariin regulates the expression of Th1- and Th17-polarizing cytokine by DCs. For this, we cultured CD4 + T cells with bone marrow-derived DCs from control and icariin-treated mice and analyzed cytokine expression. In our DC-T cell coculture system, icariin-treated DCs resulted in significantly less IFN-γ and IL-17 production from T cells (Fig. 3E). 3.3. Icariin inhibits Th1 and Th17 cell differentiation during the induction phase of EAE Next, we investigated whether a defect in inflammatory T cell development could also be detected during the induction phase of EAE. Lymph nodes, spleen and CNS of mice were harvested on day 13 after immunization with MOG35–55. Intracellular cytokine staining showed that both the proportions of Th1 and Th17 cells in the splenocytes and LN were lower in icariin-treated mice than their respective controls (Fig. 4A). The proportion of Th17 cells decreased obviously in the CNS of icariin-treated mice compared to control mice. But the proportion of Th1 cell was not changed in the CNS between the two groups (Fig. 4A). The recall response to MOG35–55 was also tested with splenocytes on day 13 EAE mice via CCK8 assays. Splenocytes exhibited diminished proliferation in icariin-treated mice (Fig. 4B). IL-17 and IFNγ productions were assayed by ELISA in culture supernatants with MOG35–55 stimulation for 72 h. Splenocytes of icariin-treated mice showed decreased production of IL-17 and IFN-γ compared to their
controls (Fig. 4C). mRNA expression of IL-17 and IFN-γ was detected by q-PCR in the two groups. Consistent with that, the genes controlling the expression of IL-17 and IFN-γ were significantly downregulated in icariin-treated mice (Fig. 4D). 3.4. Icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro Previous results showed that icarrin affected Th1 and Th17 cell proliferation in vivo; we further examined the effect of icariin on T cells in vitro. We first examined the effect of icariin on T cell proliferation. Cyclosporin A (CsA) is a potent immunosuppressive agent and has been demonstrated to have inhibitory effect on T lymphocyte proliferation. Here we used CsA as a positive control. MTT assay showed that icariin dose-dependently inhibited the proliferation of Con A-activated T cells (Fig. 5A). It is notable that icariin did not exert any significant suppressive effect on naive T cells until reaching a high dose of 30 μM (Fig. 5B), indicating a relatively low cytotoxicity. Expression of CD25 and CD69 induced by Con A is one of the hallmarks of activated T cells. Upon stimulation with Con A, T cells expressed higher levels of CD25 and CD69 compared to the control group. When treated with icariin, the percentage of CD25+ and CD69+ T cells was markedly reduced (Fig. 5C–D). Con A-activated T cells also produced an increased level of IFN-γ and IL-17A, while icariin treatment dose-dependently suppressed these cytokines (Fig. 6A). Next, we determined if icariin treatment has an effect on in vitro Th1 and Th17 cell differentiation. Naive CD4+ T cells were isolated and stimulated under Th1 and Th17 conditions for 5 days in the presence of icariin. Analysis of the IFN-γ+ and IL-17+CD4+ T-cell population revealed that icariin completely blocks the proliferation of these specific subsets (Fig. 6B–C). 4. Discussion Our current study demonstrated a crucial role for icariin in suppressing Th1 and Th17 cell differentiation and ameliorating EAE. Icariin treatment leads to alleviated inflammatory infiltration and reduces BBB leakage of the paracellular tracer. Mice that received icariin-treated T
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Fig. 3. Icariin affects inflammatory T cell development in vitro during EAE. A, in order to investigate the effect of icariin on T cell proliferation in vitro, the splenocytes harvested from mice 25 days after EAE induction were labeled with carboxyfluorescein succinimidyl amino ester (CFSE). CFSE loss by CD4+ proliferating cells from the two groups was assayed by flow cytometry after restimulation with MOG35–55 (10 mg/ml) for 72 h. The average frequency of CD4+ proliferating cells is presented; B, the harvested splenocytes were also restimulated with MOG35–55 (10 mg/ml) for 72 h, and added CCK-8 solution is incubated at 37 °C for the final 4 h. Cell proliferation was evaluated by stimulation index and the average frequency is presented; C, indicates the production of IL-17A and IFN-γ by splenocytes and LN cells from control and icariin-treated EAE mice; D, indicates genes relative expression of IL-17A and IFNγ of splenocytes and LN cells from control and icariin-treated EAE mice; and E, CD4+ T cells isolated from C57BL/6 mice were cultured with CD11c+ DCs derived from control and icariin-treated mice on day 25 after EAE induction. Supernatants from cultures were harvested 72 h after initiation of cultures and assayed by ELISA for IFN-γ and IL-17.*P b 0.05. N = 6.
cells also displayed lower EAE scores and better clinical recovery from EAE. Icariin administration suppresses the frequencies of both Th1 and Th17 cells in the splenocytes and lymph node cells. Icariin-treated mice also show lower frequency of Th17 cells in CNS mononuclear cells. Furthermore, icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. Previous studies have shown that BBB leakage breakdown is a fundamental event during the course of MS and that the magnitude of the neurovascular dysfunction in EAE is associated with the neurological severity of the disease [28]. Although the mechanisms are not completely understood, they are thought to involve disassembly of the interendothelial junctional complex and integrin focal adhesion complexes, probably as a result of altered expression of tight junction proteins in response to proinflammatory cytokine, such as TNF-α and IFN-γ [29–31]. In this study, we showed that icariin treatment plays a protective role against BBB leakage in the region of inflammation, which is contributable to the ameliorated EAE. CD4+ Th cells play an important role in the adaptive immune system by activating and directing other immune cells. There are at least four distinct Th cell subsets (Th1, Th2, Th17, and regulatory T cells) that have been demonstrated to control immune response [32]. Both Th1 and Th17 cells have been shown in clinical and preclinical studies to be critically involved in the pathogenesis of autoimmune diseases including MS, rheumatoid arthritis, and inflammatory bowel disease, whereas Th2 cells are implicated in asthma and allergy when aberrantly stimulated [33,34]. The differentiation of Th1 cells, which produce the
signature cytokine IFN-γ, depends on signaling through the IFN-γ receptor, the IL-12 receptor, and their downstream transcription factors, namely, signal transducer and activator of transcription 1 (STAT1) and STAT4. IL17-producing Th17 cells arise after IL-6 stimulation and subsequent activation of STAT3. IFN-γ-producing Th1 and IL-17-producing Th17 cells are key proinflammatory mediators of cellular immunity that underlie crucial events during development of EAE. The Th1 lineage of cytokine can help Th17 cells invade the brain and spinal thus triggering EAE [35,36]. The high percentage of Th17 cells has an impact on the inflammation in the brain and the severity of disease [37]. This study showed that the attenuation of EAE in icariin-treated mice is associated with a decrease in IL-17 and IFN-γ expression in the peripheral lymphoid organs and CNS. Based upon the study, icariin may be an effective therapeutic agent in the treatment of MS. Cytokines play an essential role in the establishment and maintenance of autoimmune disorders, such as MS and EAE [38]. T lymphocytes are characterized by IFN-γ production, which is the cytokine involved in both macrophage activation and T cell differentiation in naive CD4+ Th1 cells [39]. Furthermore, IFN-γ is related to the severity of the EAE protocol, as well as it regulates the function of T lymphocytes, playing an important role in autoimmune diseases, such as MS [40,41]. In our in vivo and in vitro experiment, the results showed suppressed expression of IFN-γ and IL-17A induced by icariin, indicating the protective role of icariin in ameliorating EAE development. Icariin showed anti-MDSC activity in the 4T1-Neu tumor-bearing mice, where treatment with icariin led to a reduction in MDSC
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Fig. 4. Icariin inhibits inflammatory T cell development during the induction phase of EAE. A, intracellular staining was used to determine the proportion of Th17 and Th1 cells in spleen, LN cells and CNS; B, splenocytes were restimulated with MOG35–55 for 72 h and the proliferation was assessed by CCK8; C, the production of IL-17A and IFN-γ in spleen and LN cells was measured by ELISA; and D; expression of IL-17A and IFN-γ mRNA in the spleen and LN cells was assessed by qPCR. *P b 0.05. N = 6.
percentages, likely due to induced differentiation toward dendritic cell [26,42]. Recently, DCs have been demonstrated as a therapeutic target in MS and EAE [27,43]. CD11c-positive DCs have been shown to be sufficient to initiate this autoimmune demyelinating disorder [44]. Further, antigen presentation by myeloid DCs has been implicated in driving progression of relapsing EAE [45]. In our current DC-T cell coculture system, icariin-treated DCs resulted in significantly less IFN-γ and IL-17
production from T cells, indicating that the effect of icariin on Th1 and Th17 cell differentiation may be mediated via modulation of DCs. In summary, our study provides evidence that icariin suppresses Th1 and Th17 cell differentiation and ameliorates EAE. Icariin treatment leads to alleviated inflammatory infiltration and reduced blood–brain barrier leakage of the paracellular tracer. Icariin administration suppresses the frequencies of both Th1 and Th17 cells in the splenocytes
Fig. 5. Icariin inhibits T lymphocyte proliferation and activation in vitro. Naive T cells from C57BL/6 mice were incubated for 72 h at 37 °C in the presence (A) or absence (B) of 5 μg/ml Con A and various concentrations of icariin or CsA. Surface CD25 (C) and CD69 (D) expressions were analyzed by flow cytometry. #P b 0.05 vs. control, *P b 0.05 vs. Con A group. N = 6.
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Fig. 6. Icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. A, naive T cells from mice were incubated for 48 h at 37 °C with 5 μg/ml Con A and various concentrations of icariin or CsA. Cell culture supernatants were collected and subjected to ELISA for cytokine analysis; B, naive CD4+ T cells were isolated and stimulated under Th1 condition in the presence of icariin for 5 days. IFN-γ+CD4+ cells were analyzed by flow cytometry; and C, naive CD4+ T cells were isolated and stimulated under Th17 condition in the presence of icariin for 5 days. IL-17+CD4+ cells were analyzed by flow cytometry. *P b 0.05 vs. control. N = 6.
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
A natural flavonoid glucoside icariin inhibits Th1 and Th17 cell differentiation and ameliorates experimental autoimmune encephalomyelitis Ruile Shen a,b,⁎, Wenjing Deng a, Chun Li a, Guangwei Zeng b a b
Department of Neurology, The First Affiliated Hospital of Zhengzhou University, China Department of Neurology, The First Affiliated Hospital of Henan University of Science and Technology, China
a r t i c l e
i n f o
Article history: Received 2 September 2014 Received in revised form 6 December 2014 Accepted 9 December 2014 Available online 17 December 2014 Keywords: Icariin Th1 cells Th17 cells EAE
a b s t r a c t Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Icariin, a natural flavonoid glucoside isolated from plants in the Epimedium family, has been proved to have various pharmacological activities. However, the effect of icariin on experimental autoimmune encephalomyelitis (EAE) has never been investigated. In our current study, we found that icariin treatment leads to alleviated inflammatory infiltration and reduced blood–brain barrier leakage (BBB) of the paracellular tracer (FITC-dextran) in EAE. Mice that received icariin-treated T cells also displayed lower EAE scores and better clinical recovery from EAE. Icariin administration suppresses the frequencies of Th1 and Th17 cells in the splenocytes and lymph node cells. Icariin-treated mice also show lower frequency of Th17 cells in CNS mononuclear cells. The effect of icariin on Th1 and Th17 cell differentiation may be mediated via modulation of dendritic cells (DCs). Furthermore, icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. In conclusion, icariin ameliorates EAE and this was associated with suppressed Th1 and Th17 cell differentiation. © 2014 Published by Elsevier B.V.
1. Introduction Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability [1]. CD4+ T cell mediated autoimmunity was suggested as one of the most important aspects of the pathogenesis [2]. Effector CD4+ T cells of Th1 and Th17 subsets are found in MS lesion and can mediate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression of Th1 and Th17 cytokines, IFN-γ and IL-17 is detected in MS lesions [3]. The role of IFN-γ and IL-17 in EAE has been extensively researched. EAE can be induced by the adoptive transfer of CNS antigen reactive Th1 cells [4–7] and Th17 cells [8–10]. Neutralization of IL-17 by treatment with anti-IL-17 antibodies has been demonstrated to reduce motor symptoms of EAE [11]. MiR-20b, as a small RNA, has been identified to suppress Th17 differentiation and the pathogenesis of EAE by targeting retinoid-related orphan receptor γt (RORγt) [12]. Other studies also demonstrated that targeting RORγt was a novel strategy for Th17 associated autoimmune diseases [13–17].
Icariin, a natural flavonoid glucoside isolated from plants in the Epimedium family, has been proved to have various pharmacological activities. Recent studies have indicated that icariin displayed positive effects on suppressing cardiac inflammation [18]. Icariin has also been demonstrated to suppress cartilage and bone degradation in a mouse model of collagen-induced arthritis [19]. However, the effect of icariin on EAE has never been investigated. In this study, we demonstrated a crucial role for icariin in suppressing Th1 and Th17 cell differentiation and ameliorating EAE. Icariin treatment leads to alleviated inflammatory infiltration and reduced BBB of the paracellular tracer (FITC-dextran) in EAE. Mice that received icariin-treated T cells also displayed lower EAE scores and better clinical recovery from EAE. Icariin administration suppresses the frequencies of both Th1 and Th17 cells in the splenocytes and lymph node cells. Icariintreated mice also show lower frequency of Th17 cells in CNS mononuclear cells. Furthermore, icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. 2. Materials and methods 2.1. Animals
⁎ Corresponding author at: Jinghua Road 24#, Jianxi District, Luoyang 471003, China. E-mail address: [email protected] (R. Shen).
http://dx.doi.org/10.1016/j.intimp.2014.12.015 1567-5769/© 2014 Published by Elsevier B.V.
Male C57BL/6 mice, all ages 6–8 weeks, were purchased from Center of Henan University of Science Technology. Mice were maintained in a
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specific pathogen-free condition. All experimental procedures were examined and approved by the Institutional Animal Care and Use Committee at Henan University of Science Technology. 2.2. Preparation of icariin Icariin (2-(4′-methoxylphenyl)-3-rhamnosido-5-hydroxyl-7glucosido-8-(3′-methyl-2-butylenyl)-4-chromanone, standard substance, HPLC N98%) was purchased from the Institute for Reference Standards and Standardization of National Institute for Food and Drug control (Beijing, China). Icariin was dissolved at a concentration of 30 mM in 100% DMSO as a stock solution, stored at − 20 °C, and diluted with a medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study (all the control groups are composed of 0.1% DMSO). 2.3. Induction of EAE Induction of EAE was conducted as described previously [20]. EAE was induced in C57BL/6 mice by immunization with 250 μg of MOG35–55 (myelin oligodendrocyte glycoprotein, MOG). All peptides were dissolved in complete Freund's adjuvant (CFA) containing 4 mg/ml of heat-killed mycobacterium tuberculosis H37Ra. At day 1 and 48 h after immunization, C57BL/6 mice were injected with 500 ng of pertussis toxin in PBS, intraperitoneally. Clinical assessment of EAE was performed after disease induction by the following criteria: 0, no disease; 1, tail paralysis; 2, hindlimb weakness or partial paralysis; 3, complete hindlimb paralysis; 4, forelimb and hindlimb paralysis; and 5, death. Icariin (25 mg/kg) was dissolved in 0.5% CMC solution and orally administered daily from day 5 to day 15 after immunization. For passive EAE experiments, donor mice were immunized with MOG/CFA in the same fashion as when inducing EAE, but no pertussis toxin was administered. Spleens and draining lymph nodes were collected 10 days later, single-cell suspensions were prepared, and RBCs were lysed. Cells (6 × 106 cells/ml) were cultured in RPMI1640 medium with 40 μg/ml of MOG35–55 peptide and 10 ng/ml of recombinant mouse IL-12 (R&D Systems). After 3 days of culture, cells were harvested and T cells were isolated by negative selection using Dynabeads (Invitrogen). Recipient mice were irradiated sublethally (500 cGy) and received 5 × 106 cells intravenously [21]. 2.4. Histopathology Mice were humanely euthanatized and spinal cord immersed in 4% paraformaldehyde for 48 h at 4 °C and paraffin embedded. Six micrometer thick (6 mm) longitudinal sections of the spinal cord were cut to include the majority of the length of the spinal cord (from cervical to lumbar regions), containing both gray and white matters. Prior to staining, sections were deparaffinized in xylene (2 × 5 min) and hydrated in graded ethanol (2 × 5 min in 100%, 5 min in 85%, 5 min 70%) to distilled water and finally rinsed in PBS. Spinal cord sections were stained with Hematoxylin/Eosin (Sigma, MO, USA) and with Luxol fast blue (American Mastertech, CA, USA) following the manufacturer's recommendations to assess cell infiltration and demyelination, respectively [22]. Four to five different sections per mouse were analyzed under light microscopy. At least 10 randomly distributed 40× fields within the white matter of the spinal cord were captured for each section. To determine the demyelination, the white matter was outlined automatically using the wand tool. The areas covered by the Luxol fast blue stain were quantified using Image-Pro Plus software (Media Cybernetics, MD, USA). The demyelination was determined as (Total white matter area − Luxol fast blue area) / Total white matter area × 100%. 2.5. In vivo BBB permeability assays On day 25 after induction of EAE, BBB permeability was determined by a published method [23]. Briefly, 70-kDa FITC-dextran (250 mg/kg)
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was injected intravenously, and 30 min later, the mice were transcardially perfused with 0.9% saline (400 ml/kg) and then with 4% paraformaldehyde (PFA, 800 ml/kg). Lumbar enlargement segments (L4–L5) were cross-sectioned at 20 μm thickness and mounted with DAPI-Fluoromount G. To determine the extravascular intensity of the FITC-dextran, images were obtained with fluorescence microscopy at ×40. The images were quantified with ImageJ software and normalized to fluorescence value in the liver and to tissue area. 2.6. Proliferative responses of T cell and cytokine analysis Spleens or draining lymph nodes were harvested and pooled from EAE mice, and single-cell suspensions were prepared. Cells were cultured at 5 × 107 cells/well in 24-well U-bottom plates with 10 mg/ml MOG35–55 peptide in complete RPMI 1640 medium. For ELISA analysis, supernatants were harvested at 72 h of culture. The concentrations of indicated cytokines were measured by quantitative capture ELISA, according to the guidelines of the manufacturers. For the detection of proliferative response, splenocytes or lymph node (LN) cells were seeded in 96-well plate at 5 × 103 cells/well with 10 mg/ml MOG35–55 peptide and then cultured for 72 h. 10 μl of CCK-8 (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan) solution was added to each well per plate, and incubated the plate at 37 °C for the final 4 h. The absorbance at 450 nm was assayed for proliferation. 2.7. Preparation and evaluation of CNS cells Brains of mice, which were perfuse with cold PBS, were dissected and incubated in 2.5 mg/ml collagenase D for 30 min at 37 °C. Singlecell suspensions were prepared. Cells were washed in RPMI 1640 medium, and mononuclear cells were isolated using a discontinuous Percoll gradient [24]. 2.8. Real time-PCR Total RNA was extracted from cultured cells or tissues using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer's instructions. mRNA levels of target genes were quantified using SYBR Green Master Mix (Takara Biotechnology, Dalian, China) with ABI PRISM 7900 Sequence Detector system (Applied Biosystems, Foster City, CA). Each reaction was performed in duplicate, and changes in relative gene expression normalized to 18sRNA levels were determined using the relative threshold cycle method. 2.9. Flow cytometry analysis Isolated mononuclear cells from spleen, LN and CNS were cultured in 24-well plates in RPMI1640 medium supplemented with 10% FBS, 200 ng/ml phorbol myristate acetate (PMA, Sigma, St. Louis, MO), and 400 ng/ml ionomycin and brefeldin A (Sigma) for 4 h. The cells were harvested and stained with FITC-anti-human CD4 at 4 °C for 30 min. After washing with PBS, the cells were fixed, permeabilized, and stained with APC-anti-IL-17 or APC-anti-IFN-γ (eBioscience, San Diego, CA) at 4 °C for 30 min. The frequencies of Th17 and Th1 cells were analyzed using a FACS cytometer equipped with CellQuest software (BD Pharmingen). 2.10. MTT cell proliferation assay Purified T cells were cultured in 96-well plates at a density of 3 × 105 cells/well in RPMI 1640 medium and stimulated with 5 μg/mol Con A in the presence of various concentrations of icariin for 72 h at 37 °C. 20 μl of MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) (4 mg/ml in RPMI 1640) was added per well 4 h before the end of incubation. MTT formazan production was dissolved by DMSO
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and the optical density at 570 nm (OD570) was measured by a microplate reader. 2.11. In vitro Th1 and Th17 cell differentiation Spleens were harvested from naive C57BL/6 mice, and single-cell suspensions were prepared. CD4+ T cells were negatively selected using the EasySep Mouse CD4+ T Cell Enrichment Kit (STEMCELL Technologies). For T-cell differentiation into Th1, or Th17, cells were labeled with anti-CD4, anti-CD44, anti-CD62L, and anti-CD25 for cell sorting. CD4+CD25−CD44loCD62L+ naive T cells were isolated by flow cytometry. Cells were stimulated with 5 μg/ml Con A under Th1 (10 ng/ml mouse IL-12, 10 μg/ml anti-mouse IL-4, 40 U/ml mouse IL-2), or Th17 conditions (2 ng/ml TGF-β, 20 ng/ml mouse IL-6, 10 μg/ml anti-mouse IL-4, 10 μg/ml anti-mouse IFN-γ, 10 ng/ml mouse IL-23) for 5 days in the presence of icariin at the indicated concentration. The frequencies of Th17 and Th1 cells were analyzed using a FACS cytometer equipped with CellQuest software (BD Pharmingen).
serum concentration of icariin is 0.78 ± 0.32 μmol/ml) decreased the EAE scores and enhanced clinical recovery from EAE (Fig. 1A). Histological analysis of spinal cord sections showed that mice treated with control PBS developed prominent inflammatory infiltration and demyelination, whereas icariin treatment showed much fewer infiltration and significantly alleviated demyelination (Fig. 1B). Next, we investigated whether icariin treatment is in part associated with a decrease in vascular leakage in the region of inflammation. We observed that the icariin treatment showed similar levels of leakage of 70-kDa FITCdextran in control mice with no EAE. After induction of EAE, the soluble marker was found to be diffused through the spinal cord parenchyma and was of high abundance in control group. In contrast to control group, the icariin treatment showed a 50% reduction in BBB leakage of the paracellular tracer (FITC-dextran) (Fig. 1C). We then transferred T cells from MOG-immunized control or icariin-treated mice into wildtype recipients. Mice that received icariin-treated T cells displayed lower EAE scores and better clinical recovery from EAE (Fig. 1D). 3.2. Icariin inhibits Th1 and Th17 cell differentiation in EAE
2.12. Statistical analysis All data were presented as means ± SEM. ANOVA analysis was used for statistical analysis with P b 0.05 being considered statistically significant. Data were analyzed using Prism software (GraphPad Software, Inc.). 3. Results 3.1. Icariin ameliorates EAE development In the present study, we used a mouse model of EAE to evaluate the therapeutic effect of icariin. Results showed that icariin treatment (with
We examined the presence of IL-17 (Th17) or interferon-γ (IFN-γ) (Th1)-producing CD4+ T cells during EAE in lymph nodes, spleen and the CNS. Lymph nodes, spleen and CNS of mice were harvested on day 25 after immunization with MOG35–55. Intracellular cytokine staining showed that the frequencies of both Th1 and Th17 cells in the splenocytes and lymph node cells were lower in icariin-treated mice than that in controls (Fig. 2A–B). Icariin-treated mice had lower frequency of Th17 cells in CNS mononuclear cells compared to respective control. But frequency of Th1 cells was not changed in the CNS between the two groups (Fig. 2C). CD4+ T cells in control-treated mice underwent cell divisions in vitro with MOG35–55, whereas CD4+ T cells in icariin-treated mice
Fig. 1. Icariin ameliorates EAE development. A, clinical scores for mice treated with icariin after the onset of EAE; B, histology of paraffin sections of spinal cords isolated from oligonucleotide-infected mice on day 25 after immunization. Spinal cord sections were stained with H&E and Luxol fast blue to assess inflammation and demyelination; C, at day 25 after induction, mice were injected intravenously with 70-kDa FITC-dextran (250 mg/kg), and lumbar spinal cords and livers were obtained after perfusion with fixative. Images of cross sections of spinal cords at the L4–L5 levels were analyzed to determine extravascular fluorescence intensity. Quantification of extravascular intensity of 70-kDa FITC-dextran. For each mouse, the fluorescence intensity of spinal cord sections was normalized to that of liver sections from the same animal; and D, MOG-primed T cells from control or icariin-treated mice were transferred into sublethally irradiated mice. The mice were scored for clinical signs of EAE and the mean clinical score was calculated by averaging the scores of all mice. Arrows indicate inflammatory cell infiltrate. *P b 0.05. N = 6.
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Fig. 2. In vivo generation of Th17 cells and Th1 cells in EAE inhibited by icariin. A, intracellular staining of IL-17 and IFN-γ in spleen cells; B, intracellular staining of IL-17 and IFN-γ in LN cells; and C, intracellular staining of IL-17 and IFN-γ in CNS infiltrating mononuclear cells. Isolated cells were fixed, permeabilized, and stained with APC-anti-IFN-γ and APC-anti-IL-17. The frequencies of Th17 and Th1 cells were analyzed using a FACS cytometer equipped with CellQuest software (BD Pharmingen). *P b 0.05. N = 6.
had a reduced proliferation (Fig. 3A). In parallel, CCK8 assays via splenocytes produced similar differences (Fig. 3B). Splenocytes and LN cells of icariin-treated mice showed decreased production of both of IL-17A and IFN-γ compared to their controls (Fig. 3C). Genes controlling the expression of IL-17A and IFN-γ were also significantly downregulated in icariin-treated mice (Fig. 3D). Myeloid-derived suppressive cells (MDSCs) are a heterogeneous group of myeloid cells comprised of hematopoietic progenitor cells and precursors of macrophages, granulocytes, and dendritic cells (DCs) [25]. Recently, icariin has been reported to exert antiinflammatory effects via modulation of MDSCs [26] and DCs have been demonstrated as a therapeutic target in MS and EAE [27]. Therefore, we tested whether icariin regulates the expression of Th1- and Th17-polarizing cytokine by DCs. For this, we cultured CD4 + T cells with bone marrow-derived DCs from control and icariin-treated mice and analyzed cytokine expression. In our DC-T cell coculture system, icariin-treated DCs resulted in significantly less IFN-γ and IL-17 production from T cells (Fig. 3E). 3.3. Icariin inhibits Th1 and Th17 cell differentiation during the induction phase of EAE Next, we investigated whether a defect in inflammatory T cell development could also be detected during the induction phase of EAE. Lymph nodes, spleen and CNS of mice were harvested on day 13 after immunization with MOG35–55. Intracellular cytokine staining showed that both the proportions of Th1 and Th17 cells in the splenocytes and LN were lower in icariin-treated mice than their respective controls (Fig. 4A). The proportion of Th17 cells decreased obviously in the CNS of icariin-treated mice compared to control mice. But the proportion of Th1 cell was not changed in the CNS between the two groups (Fig. 4A). The recall response to MOG35–55 was also tested with splenocytes on day 13 EAE mice via CCK8 assays. Splenocytes exhibited diminished proliferation in icariin-treated mice (Fig. 4B). IL-17 and IFNγ productions were assayed by ELISA in culture supernatants with MOG35–55 stimulation for 72 h. Splenocytes of icariin-treated mice showed decreased production of IL-17 and IFN-γ compared to their
controls (Fig. 4C). mRNA expression of IL-17 and IFN-γ was detected by q-PCR in the two groups. Consistent with that, the genes controlling the expression of IL-17 and IFN-γ were significantly downregulated in icariin-treated mice (Fig. 4D). 3.4. Icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro Previous results showed that icarrin affected Th1 and Th17 cell proliferation in vivo; we further examined the effect of icariin on T cells in vitro. We first examined the effect of icariin on T cell proliferation. Cyclosporin A (CsA) is a potent immunosuppressive agent and has been demonstrated to have inhibitory effect on T lymphocyte proliferation. Here we used CsA as a positive control. MTT assay showed that icariin dose-dependently inhibited the proliferation of Con A-activated T cells (Fig. 5A). It is notable that icariin did not exert any significant suppressive effect on naive T cells until reaching a high dose of 30 μM (Fig. 5B), indicating a relatively low cytotoxicity. Expression of CD25 and CD69 induced by Con A is one of the hallmarks of activated T cells. Upon stimulation with Con A, T cells expressed higher levels of CD25 and CD69 compared to the control group. When treated with icariin, the percentage of CD25+ and CD69+ T cells was markedly reduced (Fig. 5C–D). Con A-activated T cells also produced an increased level of IFN-γ and IL-17A, while icariin treatment dose-dependently suppressed these cytokines (Fig. 6A). Next, we determined if icariin treatment has an effect on in vitro Th1 and Th17 cell differentiation. Naive CD4+ T cells were isolated and stimulated under Th1 and Th17 conditions for 5 days in the presence of icariin. Analysis of the IFN-γ+ and IL-17+CD4+ T-cell population revealed that icariin completely blocks the proliferation of these specific subsets (Fig. 6B–C). 4. Discussion Our current study demonstrated a crucial role for icariin in suppressing Th1 and Th17 cell differentiation and ameliorating EAE. Icariin treatment leads to alleviated inflammatory infiltration and reduces BBB leakage of the paracellular tracer. Mice that received icariin-treated T
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Fig. 3. Icariin affects inflammatory T cell development in vitro during EAE. A, in order to investigate the effect of icariin on T cell proliferation in vitro, the splenocytes harvested from mice 25 days after EAE induction were labeled with carboxyfluorescein succinimidyl amino ester (CFSE). CFSE loss by CD4+ proliferating cells from the two groups was assayed by flow cytometry after restimulation with MOG35–55 (10 mg/ml) for 72 h. The average frequency of CD4+ proliferating cells is presented; B, the harvested splenocytes were also restimulated with MOG35–55 (10 mg/ml) for 72 h, and added CCK-8 solution is incubated at 37 °C for the final 4 h. Cell proliferation was evaluated by stimulation index and the average frequency is presented; C, indicates the production of IL-17A and IFN-γ by splenocytes and LN cells from control and icariin-treated EAE mice; D, indicates genes relative expression of IL-17A and IFNγ of splenocytes and LN cells from control and icariin-treated EAE mice; and E, CD4+ T cells isolated from C57BL/6 mice were cultured with CD11c+ DCs derived from control and icariin-treated mice on day 25 after EAE induction. Supernatants from cultures were harvested 72 h after initiation of cultures and assayed by ELISA for IFN-γ and IL-17.*P b 0.05. N = 6.
cells also displayed lower EAE scores and better clinical recovery from EAE. Icariin administration suppresses the frequencies of both Th1 and Th17 cells in the splenocytes and lymph node cells. Icariin-treated mice also show lower frequency of Th17 cells in CNS mononuclear cells. Furthermore, icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. Previous studies have shown that BBB leakage breakdown is a fundamental event during the course of MS and that the magnitude of the neurovascular dysfunction in EAE is associated with the neurological severity of the disease [28]. Although the mechanisms are not completely understood, they are thought to involve disassembly of the interendothelial junctional complex and integrin focal adhesion complexes, probably as a result of altered expression of tight junction proteins in response to proinflammatory cytokine, such as TNF-α and IFN-γ [29–31]. In this study, we showed that icariin treatment plays a protective role against BBB leakage in the region of inflammation, which is contributable to the ameliorated EAE. CD4+ Th cells play an important role in the adaptive immune system by activating and directing other immune cells. There are at least four distinct Th cell subsets (Th1, Th2, Th17, and regulatory T cells) that have been demonstrated to control immune response [32]. Both Th1 and Th17 cells have been shown in clinical and preclinical studies to be critically involved in the pathogenesis of autoimmune diseases including MS, rheumatoid arthritis, and inflammatory bowel disease, whereas Th2 cells are implicated in asthma and allergy when aberrantly stimulated [33,34]. The differentiation of Th1 cells, which produce the
signature cytokine IFN-γ, depends on signaling through the IFN-γ receptor, the IL-12 receptor, and their downstream transcription factors, namely, signal transducer and activator of transcription 1 (STAT1) and STAT4. IL17-producing Th17 cells arise after IL-6 stimulation and subsequent activation of STAT3. IFN-γ-producing Th1 and IL-17-producing Th17 cells are key proinflammatory mediators of cellular immunity that underlie crucial events during development of EAE. The Th1 lineage of cytokine can help Th17 cells invade the brain and spinal thus triggering EAE [35,36]. The high percentage of Th17 cells has an impact on the inflammation in the brain and the severity of disease [37]. This study showed that the attenuation of EAE in icariin-treated mice is associated with a decrease in IL-17 and IFN-γ expression in the peripheral lymphoid organs and CNS. Based upon the study, icariin may be an effective therapeutic agent in the treatment of MS. Cytokines play an essential role in the establishment and maintenance of autoimmune disorders, such as MS and EAE [38]. T lymphocytes are characterized by IFN-γ production, which is the cytokine involved in both macrophage activation and T cell differentiation in naive CD4+ Th1 cells [39]. Furthermore, IFN-γ is related to the severity of the EAE protocol, as well as it regulates the function of T lymphocytes, playing an important role in autoimmune diseases, such as MS [40,41]. In our in vivo and in vitro experiment, the results showed suppressed expression of IFN-γ and IL-17A induced by icariin, indicating the protective role of icariin in ameliorating EAE development. Icariin showed anti-MDSC activity in the 4T1-Neu tumor-bearing mice, where treatment with icariin led to a reduction in MDSC
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Fig. 4. Icariin inhibits inflammatory T cell development during the induction phase of EAE. A, intracellular staining was used to determine the proportion of Th17 and Th1 cells in spleen, LN cells and CNS; B, splenocytes were restimulated with MOG35–55 for 72 h and the proliferation was assessed by CCK8; C, the production of IL-17A and IFN-γ in spleen and LN cells was measured by ELISA; and D; expression of IL-17A and IFN-γ mRNA in the spleen and LN cells was assessed by qPCR. *P b 0.05. N = 6.
percentages, likely due to induced differentiation toward dendritic cell [26,42]. Recently, DCs have been demonstrated as a therapeutic target in MS and EAE [27,43]. CD11c-positive DCs have been shown to be sufficient to initiate this autoimmune demyelinating disorder [44]. Further, antigen presentation by myeloid DCs has been implicated in driving progression of relapsing EAE [45]. In our current DC-T cell coculture system, icariin-treated DCs resulted in significantly less IFN-γ and IL-17
production from T cells, indicating that the effect of icariin on Th1 and Th17 cell differentiation may be mediated via modulation of DCs. In summary, our study provides evidence that icariin suppresses Th1 and Th17 cell differentiation and ameliorates EAE. Icariin treatment leads to alleviated inflammatory infiltration and reduced blood–brain barrier leakage of the paracellular tracer. Icariin administration suppresses the frequencies of both Th1 and Th17 cells in the splenocytes
Fig. 5. Icariin inhibits T lymphocyte proliferation and activation in vitro. Naive T cells from C57BL/6 mice were incubated for 72 h at 37 °C in the presence (A) or absence (B) of 5 μg/ml Con A and various concentrations of icariin or CsA. Surface CD25 (C) and CD69 (D) expressions were analyzed by flow cytometry. #P b 0.05 vs. control, *P b 0.05 vs. Con A group. N = 6.
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Fig. 6. Icariin suppresses the proliferation of T cells and the differentiation of Th1 and Th17 cells in vitro. A, naive T cells from mice were incubated for 48 h at 37 °C with 5 μg/ml Con A and various concentrations of icariin or CsA. Cell culture supernatants were collected and subjected to ELISA for cytokine analysis; B, naive CD4+ T cells were isolated and stimulated under Th1 condition in the presence of icariin for 5 days. IFN-γ+CD4+ cells were analyzed by flow cytometry; and C, naive CD4+ T cells were isolated and stimulated under Th17 condition in the presence of icariin for 5 days. IL-17+CD4+ cells were analyzed by flow cytometry. *P b 0.05 vs. control. N = 6.
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