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Chrysin suppresses human CD14+ monocyte-derived dendritic cells and ameliorates experimental autoimmune encephalomyelitis

Journal of Neuroimmunology, November 2015, Pages 13 - 20

Abstract

Chrysin, a naturally flavonoid of plant, has various biological activities. However, the effects of chrysin on dendritic cells (DCs) and multiple sclerosis (MS) remain unknown. In this study, we demonstrate that chrysin inhibited human DC differentiation, maturation, function and the expression of the Th1 cells polarizing cytokines IFN-γ and IL-12p35 form DCs. In addition, chrysin ameliorated experimental autoimmune encephalomyelitis (EAE), an animal model of MS, by reducing CNS inflammation and demyelination. Furthermore, chrysin suppressed DCs and Th1 cells in the EAE mice. Taken together, chrysin exerts anti-inflammatory and immune suppressive effects, and suggests a possible therapeutic application of chrysin in MS.

Graphical abstract

 

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Highlights

 

  • Chrysin inhibited differentiation and endocytosis of human CD14+ monocyte-derived DCs.
  • Chrysin suppressed human DC maturation, antigen presentation and expression of cytokines for Th1 polarization.
  • Chrysin suppressed CD11c+ DC cells and Th1 cells in vivo in EAE mice.
  • Chrysin ameliorated the clinical severity and decreased the CNS inflammation and demyelination of EAE.

Abbreviations: DCs - dendritic cells, TLRs - toll-like receptors, iDCs - immature dendritic cells, mDCs - mature dendritic cells, CNS - central nervous system, PBMCs - peripheral blood mononuclear cells, GM-CSF - granulocyte-macrophage colony-stimulating factor, HLA-DR - human leukocyte antigen-DR, MHC - major histocompatibility complex, IL-12p35 - interleukin-12p35, IFN-γ - interferon-γ, LPS - lipopolysaccharide, MLR - Mixed-Lymphocyte Reaction, MS - multiple sclerosis, EAE - experimental autoimmune encephalomyelitis, MOG - myelin oligodendrocyte glycoprotein, H&E - hematoxylin–eosin.

Keywords: Chrysin, Dendritic cells, Experimental autoimmune encephalomyelitis, Multiple sclerosis, Inflammation.

1. Introduction

Dendritic cells (DCs) are professional antigen-presenting cells (APC) that play a crucial role in the initiation of immune responses (Banchereau et al, 2000 and Reis e Sousa, 2004). Bone marrow-derived DCs home to peripheral tissues and are stimulated through toll-like receptors (TLRs), at which point immature DCs (iDCs) become mature DCs (mDCs). Then, the mDCs migrate to lymphoid tissues and produce cytokines that regulate the immune cell response and function (Visintin et al, 2001, Beutler, 2004, and Macagno et al, 2007). DCs at various stages of differentiation differ in surface markers, including the costimulatory molecules CD80 and CD86 and the maturation marker CD83 as well as the human leukocyte antigen-DR (HLA-DR) in humans and the major histocompatibility complex (MHC)-II in mice. Furthermore, iDCs are very proficient at antigen uptake, whereas the main function of mDCs is to induce T cell activation. On the basis of their important roles in the immune system, DCs are a potential therapeutic target for the control of autoimmune and inflammatory disease.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS)( Noseworthy et al., 2000 ). Several immunomodulatory drugs including β-interferon and an immune modulator termed Copaxone are being introduced to treat MS. Unfortunately, the current drugs are unsatisfactory because they cause toxicity in some patients ( Rolak, 2001 ). It is therefore necessary to further improve MS therapy through the development of new drugs. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS, which is often used for studies of the pathogenesis and therapeutic interventions for this disease (Steinman and Zamvil, 2006 and Wekerle, 2008). Both MS and EAE are believed to be myelin-specific CD4+ T cell-mediated diseases, and type 1 T helper cell (Th1)-mediated EAE has been well studied. Antigens presented by APCs can stimulate naïve CD4+ T cells to differentiate into various types of effector T cells ( Zhang et al., 2015 ). More and more evidence has shown that controlling APCs including DCs can affect the development of autoimmune and inflammatory diseases (Xue et al, 2014 and Zhang et al, 2014). Thus, it is crucial to perform further studies to explore potential therapeutic drugs that could modulate DC functions.

The flavonoid chrysin is present at high levels in honey and propolis as well as in many plant extracts (Hecker et al, 1996 and Harada et al, 2003). It has been reported that chrysin has various beneficial pharmacological properties, including anti-oxidant (Bors and Saran, 1987 and Lapidot et al, 2002), anti-hypertensive ( Villar et al., 2002 ), anti-cancer ( Li et al., 2010 ) and anti-estrogenic ( Wang and Kurzer, 1998 ) effects. Reports showed that chrysin could attenuate airway inflammation by modulating Th1/Th2 polarization through suppressing the inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) (Wadibhasme et al, 2011 and Du et al, 2012). Also, chrysin pretreatment inhibited phosphorylation extracellular signal-regulated kinase (ERK), p38 and inflammatory cytokines release in mice induced with airway inflammation ( Shen et al., 2015 ). Some other studies have reported that chrysin has anti-inflammatory effects due to decreasing the production of inflammatory mediators such as prostaglandin (PG) E2, cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines (Shin et al, 2009, Ha et al, 2010, and Harasstani et al, 2010). Although chrysin has been shown to have a variety of biological effects, its effects on CNS inflammatory diseases and its mechanisms are poorly understood. In the present study, we evaluated the effects of the flavonoid chrysin on the differentiation and functions of human CD14+ monocyte-derived DCs in vitro and examined the actions of chrysin on EAE in vivo to characterize the roles and cellular mechanisms of chrysin on autoimmune CNS inflammation. The data suggested that chrysin might have the potential for clinical and therapeutic applications against MS.

2. Materials and methods

2.1. Animals

Female C57BL/6 mice (6–8 weeks) were purchased from the Academy of Military Medical Science (Beijing, China). The mice were fed and housed in a specific pathogen-free animal facility of the Experimental Animal Center of Tianjin Medical University. The experiments were in accordance with guidelines for animal care and were approved by Animal Ethics Committee of Tianjin Medical University.

2.2. CD14+ cell purification

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human blood buffy coats (Tianjin Blood Center, Tianjin, China) using Ficoll–Paque PLUS (GE Healthcare Bio-sciences AB, Uppsala, Sweden). The human CD14+ monocytes were separated from the PBMCs using a magnetic separation column according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). The purified CD14+ monocytes were analyzed using a FACS Calibur (BD Biosciences, CA, USA) to confirm the purity of CD14+ cells (> 95%). The culture medium for the primary cells was the complete RPMI 1640 culture medium (containing with 1 mM sodium pyruvate, 2 mM L-Glutamine, 100 μg/ml kanamycin and 10% fetal bovine serum (FBS)) (Gibco, Auckland, NZ).

2.3. DCs differentiation and maturation

To differentiate the DCs, the purified human CD14+ monocytes were cultured in the presence of 1000 U/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1000 U/ml interleukin-4 (IL-4) (Peprotech, Rocky Hill, USA) for 5 days. Simultaneously, the monocytes were treated with or without 5 or 25 μM of chrysin (Higher Biotech Co., Ltd., Shanghai, China). Chrysin was dissolved in DMSO (Sigma, St. Louis, USA) and diluted in cell culture medium. The final concentration of DMSO was 0.1%. Control experiments showed that this concentration had no effect on the cells. Furthermore, the effects of chrysin on the cells were not due to direct cytotoxicity as indicated by the trypan blue exclusion assay. For DCs maturation, the iDCs with or without chrysin treatment were cultured in the complete RPMI 1640 medium containing 1 μg/ml of lipopolysaccharide (LPS) (Peprotech, Rocky Hill, USA) for 2 additional days beginning on day 5.

2.4. DC surface marker expression analysis

The cells were harvested on day 5 for analysis of the differentiation of the human CD14+ monocyte-derived DCs or on day 7 for maturation analysis. DCs without added chrysin or LPS were analyzed as negative controls. The cells were labeled with fluorescently tagged anti-human DC surface marker antibodies to CD80, CD83, CD86, and HLA-DR, and simultaneously labeled with PE-conjugated CD11c antibody (BD Biosciences, CA, USA) for fluorescence-activated cell sorter (FACS) analysis. The FACS data were analyzed using Flowjo software (Tree Star, Ashland, OR).

2.5. iDC endocytosis analysis.

Endocytosis was evaluated by measuring the cellular uptake of FITC-dextran (Sigma, St. Louis, USA). The iDCs were incubated in cell medium with FITC-dextran (0.1 mg/ml), and simultaneously treated with or without 5 or 25 μM of chrysin treatment, either at 4 °C as a negative control or at 37 °C as a positive control for 30 min. The cells were then washed and analyzed using the FACS Calibur.

2.6. Mixed-lymphocyte reaction (MLR) assay

Human CD4+ T cells were selected from the PBMCs using anti-human CD4-conjugated microbeads (Miltenyi Biotec, Auburn, CA). The purified CD4+ T cells were resuspended by phosphate-buffered saline (PBS) at 107 cells/ml and treated with 2 μM CFSE (Invitrogen, Oregon, USA). LPS-activated mDCs treated with or without 5 or 25 μM chrysin were pretreated with mitomycin C (25 μg/ml) before being co-cultured with the CD4+ T cells. The CFSE-labeled CD4+ T cells and the pretreated DCs were added to the wells of a U-bottom microtiter plate (2 × 10 cells/well) at ratios of 1:10, 1:20, and 1:40 and incubated for 4 days. Finally, the cells were harvested and assayed using the FACS Calibur, and the FACS data were analyzed using Flowjo software (Tree Star, Ashland, OR).

2.7. Quantitative real-time PCR

According to the manufacturer's instructions (Invitrogen, Carlsbad, USA), total mRNA was extracted from the control group, chrysin-treated and LPS-stimulated human DC using TRIzol reagent. M-MLV reverse transcriptase (Invitrogen, Oregon, USA) was used to convert the mRNA into cDNA. Quantitative real-time PCR (qPCR) was used to determine the expression of mRNA. qPCR was performed for IFN-γand IL-12p35 using SYBR Green mix (Newbio Industry, Beijing, China). GAPDH was used as the internal control. The data are shown as 2-△Ct values and represent at least three independent experiments.

2.8. Induction, treatment and clinical assessment of EAE

To induce EAE, the mice were immunized subcutaneously in both flanks with 100 μg of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (amino acid sequence MEVGWYRSPFSRVVHLYRNGK and purity > 95%) (CL.Bio-Scientific CO., LTD, Xi'an, China). The MOG35–55 was dissolved in 100 ml of PBS and emulsified in 100 μl of Complete Freund's Adjuvant (CFA) (Sigma, St. Louis, USA) containing 500 μg of Mycobacterium tuberculosis H37RA (Difco, Detroit, USA). Immediately after immunization and again 48 h later, the mice received a tail vein injection of 200 ng of Pertussis toxin (List Biological Laboratories, CA, USA) in 100 μl of PBS. In the animal experiment, the anti-inflammatory effects of chrysin required doses of 1–100 mg/kg/day ( Bae et al., 2011 ). We observed no animal deaths using the dose of chrysin 100 mg/kg/day in experiment and there was no detectable toxicity as evaluated by weight gain and examination at necropsy. As a treatment for EAE, chrysin was administered by intragastric injection at 100 mg/kg daily for 3 days before the MOG35–55 immunization (n = 12). The mice were observed, and clinical scores were assessed daily according to a standard scoring system. The disease was graded on a scale of 0–5 of increasing severity: 0, no clinical signs; 1, tail paralysis; 2, one hindlimb paralysis; 3, complete hindlimb paralysis; 4, hindlimb and forelimb paralysis; 5, moribund/death.

2.9. Histology and immunofluorescence analysis

The lumbar spinal cords were harvested from the EAE mice on day 15 post-immunization. Subsequently, these were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into sections 12 μm in thickness. The paraffin sections of lumbar spinal cords were stained with hematoxylin–eosin (H&E) for analysis of inflammatory cell infiltration and with Luxol fast blue (Alfa Aesar, Ward Hill, USA) to evaluate the demyelination. The immunofluorescence, paraffin sections were immunostained with anti-CD4 (eBioscience, San Diego, CA) and anti-F4/80 (eBioscience, San Diego, CA) for detecting CD4 positive T cells and macrophages infiltration respectively.

2.10. Intracellular cytokine staining

Single cell suspensions of the lymphocytes were prepared from the PBS- or chrysin-treated EAE mice on day 15 post-immunization by mashing and passing through a cell strainer. Then, the lymphocytes were re-stimulated with 1 μg/ml brefeldin A (Sigma, St. Louis, USA), 600 ng/ml phorbol 12-myristate 13-acetate (PMA) (Enzo Life Sciences, Farmingdale, USA) and 1 μg/ml ionomycin (Enzo Life Sciences, Farmingdale, USA). After 5 h, the lymphocytes were collected and labeled with APC-conjugated rat anti-mouse CD4 antibody (eBioscience, San Diego, CA). Then, the cells were fixed and permeabilized for intracellular cytokine staining with PE-conjugated rat anti-mouse IFN-γ antibody (eBioscience, San Diego, CA). Nonspecific staining was monitored using isotype antibody controls. Subsequently, the cells were analyzed using the FACS Calibur, and the acquired data were analyzed with CellQuest software and Flowjo software (Tree Star, Ashland, OR).

2.11. 2DC surface marker expression analysis in spleen

Single cell suspensions of the splenocytes were prepared from the PBS- or chrysin-treated EAE mice on day 15 post-immunization. The cells were re-stimulated with MOG35–55 (20 μg/ml) for 48 h and then collected. Expression of CD11c, CD83, CD86, MHCII was analyzed by staining with fluorochrome-conjugated specific mAbs or isotype controls (Sungene, China). The labeled cells were analyzed using the FACS Calibur, and the acquired data were analyzed with CellQuest software and Flowjo software (Tree Star, Ashland, OR).

2.12. Statistical analysis

The data from the treated and control groups were compared using Student's t-test. The differences were considered to be significant when p < 0.05. The standard deviation from the mean was also calculated and expressed as SD values.

3. Results

3.1. Chrysin inhibited differentiation and endocytosis of human iDCs

To explore the effect of chrysin on DC differentiation, purified human CD14+ monocytes were allowed to differentiate into iDCs in the presence of 5 or 25 μM chrysin. After 5 days, the expression of the surface markers on the DCs was assessed using FACS. Our study showed that chrysin significantly reduced the expression of the maturation marker CD83 and the costimulatory molecules CD86 and HLA-DR in a concentration-dependent manner, while the expression of CD80 showed little difference among three groups. Data presented as the mean fluorescence intensity (MFI) and as the percentage of the values for the PBS-treated DC controls ( Figs. 1 A, B). These results indicated that chrysin suppressed the DC differentiation from human CD14+ monocytes. Furthermore, our study determined whether chrysin could affect the receptor-mediated endocytic function of iDCs. The antigen-uptake capacity of iDCs was determined by measuring the cellular uptake of FITC-dextran. As shown in Fig. 1 C, the FITC-dextran uptake was decreased in the chrysin-treated iDCs in a dose-dependent manner. These data suggested that chrysin could inhibit the differentiation and the function of iDCs.

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Fig. 1 Chrysin inhibited differentiation and endocytosis of human DCs. Purified human CD14+ monocytes were cultured and treated with or without 5 or 25 μM of chrysin for 5 days. To assess the differentiation, the cell surface markers CD80, CD83, CD86 and HLA-DR were examined by FACS. (A) Histograms of the expression of the cell surface markers. The x-axis represents the FITC-labeled DC marker staining. The numbers indicate the MFI of the stained cells. (B) The relative levels of the cell markers compared to the PBS-treated cells (100%). The MFI level for each chrysin treatment was compared to that of the controls to calculate the percentage of the controls. To assess the endocytosis, dextran-FITC uptake by the iDCs was evaluated by FACS. (C) The results of the dextran-FITC uptake. a. 37 °C positive control: PBS-treated iDCs, 37 °C; b. 5 μM chrysin-treated iDCs, 37 °C; c. 25 μM chrysin-treated iDCs, 37 °C; d. 4 °C control: PBS-treated iDCs, 4 °C for 30 min as a negative control. These data represent one of three independent experiments with different blood donors.

3.2. Chrysin suppressed DC maturation, antigen presentation and expression of Th1-polarizing cytokines

DCs play a significant role in adaptive immunity, and only mDCs can present antigenic peptides to naïve T cells. Therefore, it was important to explore the effects of chrysin on the phenotypic and functional maturation of mDCs. iDCs were activated to become mDCs by treatment with LPS for 48 h. The surface marker expression on the mDCs was examined by FACS. Chrysin clearly reduced the LPS-activated expression of CD80, CD83, CD86 and HLA-DR compared with the PBS-treated DCs ( Fig. 2 A, B). These results showed that chrysin inhibited the LPS-induced DC maturation.

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Fig. 2 Chrysin suppressed the LPS-induced DC maturation, antigen presentation and the expression of cytokines associated with the mDC-induced Th1 polarization. iDCs were treated with LPS at day 5 and cultured for 2 additional days to induce DCs maturation. To assess maturation, the cells were stained with FITC-conjugated antibodies to DC surface markers CD80, CD83, CD86 and HLA-DR and analyzed by FACS. (A) Expression of mDC surface markers. Numbers indicate the MFI for the antibody staining. (B) The relative levels of the cell markers compared to the PBS-treated controls (100%). The MFI level for each chrysin treatment was normalized to that of controls to calculate the percentage of the controls. (C) To evaluate antigen presentation, the mDCs were treated with Mitomycin C (25 μg/ml) and the CD4+ T cells were treated with CFSE (2 μM). Then, the mDCs were co-cultured with CD4+ T cells at ratios of 1: 10, 1: 20 and 1: 40 for 4 days. FACS was performed to detect allogeneic T cell proliferation. (D) iDCs pretreated with or without chrysin for 2 h were stimulated with or without LPS for 8 h. The mRNA was extracted, and the expression levels of selected genes were measured by qPCR. Relative mRNA expression of IFN-γ and IL-12p35. The data are expressed as the means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test). These data represent one of three independent experiments with different blood donors.

To further investigate the effect of chrysin on antigen presentation by the mDCs, allogeneic T cells were stimulated by mDCs treated with or without chrysin. T cell proliferation was assessed using the MLR. Chrysin decreased the level of allogeneic T cell proliferation in a dose-dependent manner at all of DC-to-T cell ratios tested ( Fig. 2 C). The data suggested that chrysin suppressed the mDCs-induced allogeneic T cell proliferation.

Cytokines produced by DCs play an important role in the activation of T cells. Therefore, our study further explored the effect of chrysin on the mRNA expression of the cytokines associated with the Th cell polarization induced by the DCs. Human DC were pretreated with either chrysin or PBS for 2 h and then were activated by LPS (1 μg/ml) for 8 h before harvesting. The mRNA expression of the cytokines involved in Th cell polarization was detected using real-time reverse-transcription polymerase chain reaction (qRT-PCR). The data demonstrated that the mRNA expression of the Th1-associated cytokine IFN-γ and Th1 cells polarizing cytokine IL-12p35 were suppressed in the DCs treated with chrysin and LPS compared with those treated with PBS and LPS ( Fig. 2 D). However, the expression levels of IL-6, IL-4, TNF-α and IL-23, which are involved in Th17 cell and Th2 cell polarization, were little changed among the different groups (data not shown). Based on these results, chrysin might suppress Th1 polarization.

3.3. Chrysin ameliorated the clinical severity of EAE

To further explore the immunosuppressive role of chrysin in vivo, the Th1 and Th17 cell-mediated autoimmune inflammatory disease EAE was induced by MOG in C57BL/6 mice. Chrysin was administered by intragastric injection daily until mice were sacrificed on day 35 post-immunization. The results showed that the incidence of EAE was not different between the PBS- and chrysin-treated mice ( Fig. 3 A). However, the disease onset for the chrysin-treated mice was delayed 6 days compared with the PBS-treated mice. Furthermore, the overall health condition of chrysin-treated mice was much better than PBS-treated mice. The data showed that chrysin significantly decreased the mean clinical score for the EAE ( Fig. 3 B). Furthermore, the cumulative disease score was distinctly suppressed in the chrysin-treated mice (39.00 ± 8.76 vs 65.08 ± 9.41, p < 0.001) ( Fig. 3 C). Finally, the maximal disease score was also obviously decreased in the chrysin-treated mice compared with the PBS-treated mice (2.83 ± 0.26 vs 3.50 ± 0.32, p < 0.01) ( Fig. 3 D). In summary, these data indicated that development and progression of EAE were ameliorated by chrysin.

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Fig. 3 Chrysin ameliorated the clinical severity of EAE. EAE was induced with MOG, and the mice were treated with chrysin or PBS (n = 12). (A) The disease incidence is represented by the percentage of mice suffering from EAE. Mice with a clinical score ≥ 1 for two consecutive days were considered to exhibit EAE. (B) Mean of the daily clinical scores are reported for day 1 to day 35 post-immunization (day 9–17 and day 26–35, p < 0.05). (C) The mean disease cumulative score of the sum of daily clinical scores were observed from day 1 to 35 post-immunization. (D) The mean maximum clinical scores were calculated from the maximum clinical scores of individual animals from day 1 to 35 post-immunization. The values represent the means ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test). The data are representative of at least two experiments with similar results.

3.4. Chrysin decreased the CNS inflammation and demyelination of EAE

To corroborate the clinical results, we performed histological analysis of the lumbar spinal cords at the peak of the acute phase of EAE. Paraffin sections of the lumbar spinal cords were stained with H&E for analysis of the inflammatory infiltration and with Luxol fast blue for evaluation of the demyelination. The results of the H&E staining demonstrated that smaller numbers of inflammatory cells were present in the white matter of the lumbar spinal cords of the chrysin-treated mice ( Fig. 4 A). Staining with Luxol fast blue revealed that the PBS-treated mice exhibited large areas of demyelination, but the demyelination was markedly reduced in the chrysin-treated mice ( Fig. 4 B). Furthermore, CD4+ lymphocytes and macrophages were decreased in the chrysin-treated mice ( Fig. 4 C, D). Overall, chrysin inhibited the CNS inflammation and demyelination in EAE, suggesting that chrysin improved the histopathological events of EAE.

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Fig. 4 Chrysin improved the CNS inflammation and demyelination in EAE mice. Serial sections of lumbar spinal cord were obtained from the EAE mice on day 15 after immunization. (A) The H&E staining shows infiltration of inflammatory cells into the white matter of the lumbar spinal cords. The arrows indicate infiltration of inflammatory cells. (B) Luxol fast blue staining shows areas of intact myelin (blue) and demyelination (pink). The arrows indicate demyelination in the lumbar spinal cord, which is associated with infiltration of inflammatory cells. (C, D) Paraffin sections of the lumbar spinal cords were immunostained with FITC-CD4 for CD4 positive T cell and FITC-F4/80 for macrophages.

3.5. Chrysin suppressed expression of the CD11c+ cell and Th1 cell in splenocytes

The results described above suggested that chrysin significantly inhibited the expression of the surface molecules of human CD14+ monocyte-derived DCs and thereby the suppressed functions of DCs in vitro. To evaluate these effects in vivo, our study explored the potential role of chrysin on the expression of selected DC surface molecules in the EAE mice. Single cell splenocyte suspensions were prepared from PBS- or chrysin-treated EAE mice. The splenocytes were analyzed for the expression of CD83, CD86 and MHCII. As shown, the surface expression of CD83, CD86 and MHCII was decreased in the CD11c+ cells of the chrysin-treated mice ( Figs. 5 A, B, C). These results showed that chrysin suppressed the expression of the surface molecules on the murine DCs, similar to the effect of chrysin on the human CD14+ monocyte-derived dendritic cells.

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Fig. 5 Chrysin suppressed DCs and Th1 cells in mice. Suspensions of single splenocytes were isolated from the spleens of the PBS- or chrysin-treated mice on day 15 after immunization. (A) The cells were stained with CD11c and CD83 antibodies after MOG stimulation and assayed by FACS. (B) The cells were stained with CD11c and CD86 antibodies after MOG35–55 stimulation and assayed by FACS. (C) The cells were stained with CD11c and MHCII antibodies after MOG35–55 stimulation and assayed by FACS. Suspensions of single lymphocytes were isolated from the lymph nodes on day 15 after immunization. The cells were stained with CD4 and IFN-γ antibodies using intracellular cytokine staining. Then, the cells were assayed by FACS. (D) Percentage of IFN-γ-producing CD4+ lymphocytes. The values represent the means ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test). The data are representative of at least two experiments with similar results.

Because EAE is mediated by inflammatory Th cells, our study further explored the impact of chrysin on the Th cell subsets in the lymph nodes using intracellular staining. Lymph node cells were acquired from the two groups of EAE mice. The results showed that the percentage of IFN-γ-producing CD4+ lymphocytes was lower in chrysin-treated mice ( Fig. 5 D), but the percentage of IL-17-producing CD4+ lymphocyte cells was not significant changed in the chrysin-treated mice compared with the PBS-treated mice (data not shown). These results showed that the chrysin-induced suppression of the EAE was associated with a decrease in the IFN-γ-producing Th1 cells.

4. Discussion

Inflammatory processes are orchestrated by inflammatory cells and inflammatory mediators through a complex set of chemical signals. DCs play a crucial role in the initiation of immune responses (Banchereau et al, 2000 and Reis e Sousa, 2004). mDCs present antigenic peptides to naïve CD4+ T cells, and the naïve T cells subsequently differentiate into various types of Th cells that initiate the immune responses (Visintin et al, 2001, Beutler, 2004, and Macagno et al, 2007). Antigen presentation lies at the gateway to adaptive immunity and requires the involvement of the major histocompatibility complex (MHC) as well as costimulatory signals. The human MHC is called human leukocyte antigen (HLA). According to the literature, MS patients have upregulated costimulatory molecules and MHC (Windhagen et al, 1995 and Sercarz, 2000). Human CD14+ monocytes were isolated from the PBMCs, and the purified CD14+ monocytes were allowed to differentiate into DCs in presence of GM-CSF and IL-4 ( Sallusto and Lanzavecchia, 1994 ). Human DCs derived in this manner from peripheral blood mononuclear cells are widely used to study the effect of different therapeutic agents on the surface marker expression and functions of iDC and mDC (Xue et al, 2014 and Zhang et al, 2014).

A majority of studies have reported that chrysin has anti-inflammatory effects on monocytes/macrophages (Hecker et al, 1996, Liang et al, 2001, and Hougee et al, 2005), mast cell-mediated allergic inflammation ( Bae et al., 2011 ) and inflammatory bowel diseases ( Shin et al., 2009 ). However, the effect of chrysin on human CD14+ monocyte-derived DCs and CNS inflammatory diseases remains unknown. In this study, we showed that chrysin suppressed the differentiation, maturation, and antigen uptake of human CD14+ monocyte-derived DCs, and also inhibited mDCs' stimulatory effects on allogeneic T cell proliferation in vitro. On the basis of these studies, we further tested the effect of chrysin in vivo. The results demonstrated that chrysin significantly ameliorated the pathogenic Th cell-mediated EAE by reducing the CNS inflammation and demyelination. Furthermore, chrysin suppressed the expression of the costimulatory molecules of the DCs in the EAE mice. Finally, the data indicated an inhibitory role for the flavonoid chrysin in the development of the DCs as well as in neuroimmune inflammatory diseases.

The present study showed that chrysin has profound effects on human DCs during the process of differentiation from monocytes. Our results were consistent with previous reports, which reported that the monocytes in the PBMC were specifically eliminated by chrysin in vitro and that chrysin dose-dependently inhibited both the proinflammatory cytokine production and metabolic activity of LPS-stimulated PBMC ( Hougee et al., 2005 ). Consistently, our study demonstrated that chrysin-induced phenotypic and functional changes in DCs. The trypan blue exclusion assay clearly showed that the effect of chrysin on the human CD14+ monocyte-derived dendritic cells was not due to a cytotoxic effect on the monocytes/DCs. In addition, we first evaluated whether chrysin inhibited the receptor-mediated endocytic function of iDCs and the stimulation of allogeneic T cell proliferation by the mDCs. Our results showed that the chrysin-treated mDCs had an impaired potential to induce T cell proliferation and a decreased expression of HLA-DR molecules and costimulatory molecules. Thus, the inhibitory effects of chrysin on the modulation of antigen presentation were important for alleviating the pathogenesis of MS and EAE.

Th1 and Th17 cells are key proinflammatory cells in cellular immunity that underlie crucial pathological events during development of EAE. In MS patients, elevated levels of IFN-γ and IL-17 correlate with exacerbations of the disease (Windhagen et al, 1995, Diveu et al, 2008, and Kebir et al, 2009). In this report, we first demonstrated that chrysin could attenuate the disease severity by reducing the inflammatory cell infiltration and demyelination in spinal cords and by decreasing the numbers of IFN-γ-producing cells. However, IL-17A-producing cells were detected as well, and little difference was found between the chrysin-treated group and the PBS control group. DCs are considered to be the main APC population and to participate in the regulation of T cell responses in CNS ( Almolda et al., 2011 ). Cytokines produced by the DCs are important in promoting the differentiation of Th1 and Th17 cells (Ponichtera and Stadecker, 2015 and Schinnerling et al, 2015). Interleukin-12 (IL-12) stimulates differentiation of naïve CD4 T (Th0) cells into Th1 cells. In addition, the differentiation of murine Th17 cells from naïve CD4 T cells requires the concomitant activity of IL-6 and transforming growth factor-β (TGF-β) (Mangan et al, 2006, Veldhoen et al, 2006, and Zhang et al, 2006). TNF-α and IL-6 are also proinflammatory cytokines involved in several inflammatory diseases. The previous study reported that chrysin could decrease expression of target genes including TNF-α, IL-6 and IL-12 ( Lee et al., 2009 ). On the basis of our research showing that chrysin inhibited human CD14+ monocyte-derived DCs, the effects of chrysin to ameliorate EAE may be due to its prevention of the function of DCs. Therefore, our study further explored the inhibitory effect of chrysin on the DC expression of cytokines involved in the Th1 and Th17 responses. As shown in Fig. 2 D, chrysin could suppress the mRNA expression of IFN-γ and IL-12p35, which are associated with the Th1 cell differentiation and response, by the mDCs. However, the mDCs' expression of mRNA of cytokines involved in the Th17 response was not inhibited by chrysin (data not shown). Previous studies have demonstrated that regulation of excessive microglia activation may be therapeutic in neurodegenerative diseases ( Ransohoff and Perry, 2009 ). One study showed that chrysin suppresses the LPS-stimulated proinflammatory responses in microglial cells ( Ha et al., 2010 ). These results suggest that the ability of chrysin to inhibit proinflammatory cells could be useful in the treatment of neuroinflammatory diseases.

Many reports have shown that chrysin exhibits strong inflammatory suppressive properties though various signaling pathways (Bae et al, 2011 and Rehman et al, 2013). However, it is not known which pathway is involved in the chrysin-mediated suppression of DC differentiation, maturation, function and expression of the cytokines involved in the Th1 cell response. Therefore, in a future study, we will further explore the potential molecular mechanisms by which chrysin exert its immunomodulatory effects on autoimmune inflammation.

Taken together, it is clear that chrysin holds great promise as a therapy for inflammatory diseases. Although the anti-inflammatory effects of chrysin in several diseases have been studied, the roles of chrysin in human CD14+ monocyte-derived dendritic cells and the EAE system are reported here for the first time. These results suggest a possible therapeutic application of chrysin in autoimmune inflammatory diseases. In future research, the underlying molecular mechanisms of chrysin need to be explored.

5. Conclusions

These results indicated that chrysin suppressed the human DCs differentiation, maturation and antigen presentation. In addition, chrysin restricted the expression of cytokines for Th1 polarization in DCs. Chrysin alleviated the clinical symptoms of mice EAE and suppressed DCs and Th1 cells in vivo in EAE mice. Our findings suggested that chrysin may have anti-inflammatory and immunosuppressive properties in mice EAE.

Acknowledgments

This work is supported by Ministry of Science and Technology of China through Grant No. 2012CB932503 and 2011CB933100; National Natural Science Foundation of China through Grant No. 81172864, 81272317, 81170443, 81302568, 81301026; Natural Science Foundation of Tianjin through Grant No. 14JCTPJC00487 and grant No. TD12-5025 from Tianjin city.

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Footnotes

a Laboratory of Immunology and Inflammation, Department of Immunology and Research Center of Basic Medical Sciences, Basic Medical College, Medical University, Tianjin 300070, China

b Tianjin Key Laboratory of Cellular and Molecular Immunology, Tianjin Medical University, Tianjin 300070, China

c Key Laboratory of Educational Ministry of China, Tianjin Medical University, Tianjin 300070, China

d Key Laboratory of Hormones and Development, Ministry of Health, Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China

e Department of Immunology and Transplantation, Tianjin First Central Hospital, Tianjin 300192, China

Correspondence to: Department of Immunology and Research Center of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China.

⁎⁎ Correspondence to: Department of Immunology, Basic Medical College, Tianjin Medical University, Tianjin 300070, China.

1 These authors contributed equally to this work.


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