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Inhibition of bone marrow-derived dendritic cell maturation by glabridin
International Immunopharmacology 10 (2010) 1185–1193
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Inhibition of bone marrow-derived dendritic cell maturation by glabridin Jee Youn Kim a,1, Jong Soon Kang b,1, Hwan Mook Kim b, Hwa Sun Ryu a, Hyung Sook Kim a, Hong Kyung Lee a, Yeon Jin Kim a, Jin Tae Hong a, Youngsoo Kim a, Sang-Bae Han a,⁎ a b
College of Pharmacy and Medical Research Center (CICT), Chungbuk National University, 410 Seongbong, Heungduk, Cheongju, Chungbuk 361-763, Republic of Korea Korea Research Institute of Bioscience and Biotechnology, Ochang, Chungbuk 363-883, Republic of Korea
a r t i c l e
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Article history: Received 21 January 2010 Received in revised form 23 June 2010 Accepted 24 June 2010 Keywords: Glabridin Dendritic cells Maturation NF-κB MAPKs
a b s t r a c t Glabridin has multiple pharmacological activities including anti-microbial, anti-atherosclerotic, anti-nephritic, anti-inflammatory and cardiovascular protective activities. In this study, we investigated the effect of glabridin on dendritic cells, which play an essential role in innate and adaptive immune responses. Glabridin inhibited lipopolysaccharide-, poly (I:C)-, or zymosan-induced phenotypic maturation of dendritic cells (DCs), as proven by the decreased expression of CD40, CD80, CD86, MHC-I, and MHC-II. Glabridin decreased the functional maturation of DCs, in that glabridin attenuated pro-inflammatory cytokine production of IL-12, IL-1β, TNF-α, and IFN-α/β, enhanced antigen capture capacity, inhibited migration to SDF-1α and MIP-3β, and impaired induction of allogenic T cell activation. We also showed that glabridin inhibited zymosan-induced inflammation in mice. As a mode of action, we showed that glabridin inhibited degradation of IκΒα/β, nuclear translocation of NF-κB p65/ p50, and phosphorylation of ERK, JNK, and p38 MAPKs. Taken together, the present results show that glabridin inhibits dendritic cell maturation by blocking NF-κB and MAPK signalings. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Dendritic cells (DCs) are potent antigen-presenting cells that play a pivotal role in the innate and adaptive immune responses [1]. DCs originate from bone marrow precursors and migrate to almost every tissue, where they reside in an immature state [2]. Immature DCs are equipped to capture antigens and to produce large numbers of immunogenic MHC–peptide complexes. In the presence of maturation-inducing stimuli, such as inflammatory cytokines, or stimulation via CD40 ligand or toll-like receptor ligands (TLRs), including lipopolysaccharide (LPS), DCs undergo maturation with up-regulation of adhesion and co-stimulatory molecules. Mature DCs migrate to secondary lymphoid tissues and present antigen to naïve T cells and initiate T cell responses [3]. Since DCs play a key role in the initiation of the immune response, they are considered promising tools and targets for immunotherapy in several immune diseases [4]. The root of Glycyrrhiza glabra (licorice) has long been used as an antidote, demulcent, expectorant, and remedy for allergic inflammation [5]. Licorice contains glycyrrhizin, oleane triterpenoids, glucose and several flavonoids [6]. As a polyphenolic flavonoid and a major component of the hydrophobic fraction of licorice extract, glabridin, (R)-4-(3,4-dihydro-8,8-dimethyl-2H,8H-benzo[1,2-b:3,4-b′]dipyran3yl)-1,3-benzenediol, is known to possess multiple pharmacological activities. Glabridin was initially identified as an anti-microbial agent, ⁎ Corresponding author. Tel./fax: +82 43 261 2815. E-mail address: [email protected] (S.-B. Han). 1 These authors contributed equally to this work. 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.06.025
and later as an anti-melanogenic, anti-nephritic, cardiovascular protective, anti-obesity, anti-ulcer, and anti-fungal agent [6–11]. In addition, glabridin was reported to have anti-atherosclerotic activity through its inhibition of low-density lipoprotein oxidation [12]. Glabridin enhanced the efficacy of cancer chemotherapy by blocking P-glycoprotein and multidrug resistant protein 1 [13]. Interestingly, glabridin exhibited estrogen receptor agonism and growth inhibitory action in breast cancer cells [14]. Glabridin had a neuroprotective effect via modulation of neuronal cell apoptosis [15]. Among the multiple pharmacological activities of glabridin, we focused on its anti-inflammatory activity. Until now, macrophages were the only target cells of glabridin with regard to its antiinflammatory activity [5]. In this study, we investigated the suppressive activity of glabridin on other target immune cells, especially DCs, which play an important role in innate and adaptive immunity. We demonstrated that glabridin inhibited DC maturation by blocking NF-κB and MAPK signaling pathways. 2. Materials and methods 2.1. Materials Glabridin was purchased from Wako Pure Chemicals (97% purity, catalogue number 070-04821, Osaka, Japan). It was dissolved in dimethyl sulfoxide and stored at −20 °C. Female C57BL/6 (H-2b) and BALB/c (H-2d) mice (6–8 weeks old) were obtained from Korea Research Institute of Bioscience and Biotechnology (Chungbuk, Korea). Mice were housed in specific pathogen-free conditions at 21–
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24 °C and 40–60% relative humidity under a 12-h light/dark cycle. All animals were acclimatized for at least 1 week prior to the experiments. The experimental procedures used in this study were approved by the CBNU Animal Experimentation Ethics Committee. Anti-mouse antibodies against CD11c, CD40, CD80, CD86, MHC-I and MHC-II were purchased from BD Biosciences (San Jose, CA, USA) and ERK, p38, and JNK were purchased from Cell Signaling (Danvers, MA, USA). 2.2. Generation of bone marrow-derived DCs DCs were generated from bone marrow (BM) cells obtained from 6–7-week-old female mice [16]. Briefly, BM cells were flushed out from femurs and tibias. After red blood cells were lysed, whole BM cells (2 × 105 cells/ml) were cultured in 100-mm2 culture dishes in 10 ml/dish of complete medium containing 2 ng/ml GM-CSF (R&D Systems, Minneapolis, MN, USA). On day 3, another 10 ml of fresh complete medium containing 2 ng/ml GM-CSF was added, and half of the medium was changed on day 6. On day 8, non-adherent and loosely adherent DCs were harvested by vigorous pipetting and used as immature DCs (iDCs). iDCs recovered from these cultures were generally N85% CD11c+, but not CD3+ and B220+. 2.3. Phenotype analysis Phenotypic maturation of DCs was analyzed by flow cytometry [17]. Cell staining was performed using a combination of FITCconjugated anti-CD40, anti-CD80, anti-CD86, or anti-MHC plus PEconjugated CD11c antibodies. Cells were analyzed using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using WinMdi software (Scripps, La Jolla, CA, USA). Forward and side scatter parameters were used to gate live cells. Cell viability was examined by the propidium iodide (PI) nuclear staining method. Cells were stained with 1 μg/ml of PI and analyzed with a FACSCanto flow cytometer. Cells stained with PI were considered dead cells [18].
G-3′, antisense, 5′-TGA CGT CAT AGG CAA TGT TGA GCT-3′; and β-actin, sense, 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′, antisense 5′-TAA AAC GCA GCT CAG TAACAG TCC G-3′. PCR products were fractionated on 1% agarose gels and stained with 5 μg/ml ethidium bromide [17]. 2.7. Western blots Cell lysates were prepared as previously described [17]. Detergentinsoluble materials were removed, and equal amounts of protein were fractionated by 10% SDS-PAGE and transferred to pure nitrocellulose membranes. Membranes were blocked with 5% BSA in Tween 20 plus TBS (TTBS) for 1 h and then incubated with an appropriate dilution of primary antibody in 5% BSA (in TTBS) for 2 h. Blots were incubated with biotinylated antibody for 1 h and further incubated with streptavidin conjugated to HRP for 1 h. Signals were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.8. Mixed lymphocyte reaction (MLR) Responder T cells were purified from the spleen of BALB/c mice by negative depletion using biotinylated antibodies for B220, GR-1, and CD11c (BD Pharmingen, San Diego, CA, USA) and Dynabeads M-280 streptavidin (Invitrogen, Dynal, Carlsbad, CA, USA), as previously described [19]. Purity was typically more than 90%. DCs generated from the BM cells of C57BL/6 mice were treated with 40 μg/ml mitomycin C (MMC) for 1 h. One million MMC-treated DCs were added to 1 × 105 T cells in U-bottom 96-well plates (activator:responder cell ratio = 10). Allogenic T cells were pulsed with [3H]-thymidine (113 Ci/nmol, NEN, Boston, MA, USA) at a concentration of 1 μCi/well for the last 18 h and then harvested on day 5 using an automated cell harvester (Innotech, Dottikon, Switzerland). The amount of [3H]thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac, Turku, Finland) [16]. 2.9. Zymosan-induced inflammation
2.4. Endocytosis assay To analyze the endocytosis of DCs, 4 × 105 DCs were incubated at 37 °C for 1 h with 0.7 mg/ml FITC-dextran (42,000 Da, Sigma-Aldrich, St. Louis, MO, USA). After incubation, cells were washed twice with cold washing buffer (PBS containing 0.5% BSA) and stained using PEconjugated anti-CD11c antibody. Double-stained DCs were analyzed by flow cytometry. In addition, parallel experiments were performed at 4 °C to determine the nonspecific binding of FITC-dextran to DCs [17].
Inflammation was induced by subcutaneous injection of 50 μl of 3 mg/ ml zymosan A isolated from Saccharomyces cereviciae (Sigma-Aldrich, St. Louis, MO, USA) into the hind footpad of C57BL/6 mice. Glabridin was intraperitoneally administered from day 0 to day 5. On day 6, right and left popliteal lymph nodes (pLNs) were isolated and weighed [20]. 2.10. Chemotaxis
Levels of IL-12p70, IFN-γ and IL-4 in culture supernatants were measured using commercial immunoassay kits (R&D Systems, Minneapolis, MN, USA).
Chemotaxis assays were performed by using a transwell chamber as previously described [19]. DCs were added in a volume of 100 μl to the upper wells of a 24-well transwell plate with a 5 μm insert. Lower wells contained various doses of chemokines in 600 μl complete RPMI 1640 medium. The number of cells that migrated to the lower well following a 2 h incubation was counted by using a flow cytometer.
2.6. Reverse transcription-polymerase chain reaction (RT-PCR)
2.11. Statistics
Total RNA was isolated using TRIZOL™ Reagent (Molecular Research Center, Cincinnati, OH, USA). For RT-PCR, single-strand cDNA was synthesized from 2 μg total RNA. The primer sequences used were as follows: IL-12p40, sense, 5′-AGA GGT GGA CTG GAC TCC CGA-3′, antisense, 5′-TTT GGT GCT TCA CAC TTC AG-3′; TNF-α, sense, 5′-AGG TTC TGT CCC TTT CAC TCA CTG-3′, antisense, 5′-AGA GAA CCT GGG AGT CAA GGT A-3′; IL-1β, sense, 5′-ATG GCA ATG TTC CTG AAC TCA ACT-3′, antisense, 5′-CAG GAC AGG TAT AGA TTC TTT CCT TT-3′; IFN-α, sense, 5′TCT GAT GCA GCA GGT GGG-3′, antisense, 5′-AGG GCT CTC CAG AYT TCT GCT CTG-3′; IFN-β, sense, 5′-CCA CAG CCC TCT CCA TCA ACT ATA AGC-3′, antisense, 5′-AGC TCT TCA ACT GGA GAG CAG TTG AGG-3′; CXCR4, sense, 5′-GGC TGT AGA GCG AGT GTT GC-3′, antisense, 5′-GTA GAG GTT GAC AGT GTA GAT-3′; CCR7, sense, 5′-ACA GCG GCC TCC AGA AGA ACA GCG
Data represent the mean +/− STD of more than three samples and all experiments were performed more than three times. Standard deviations (STD) were calculated using the Student's t test and p values were calculated using ANOVA software (GraphPad Software, San Diego, CA, USA).
2.5. Measurement of secreted IL-12p70, IFN-γ and IL-4
3. Results 3.1. Glabridin blocks the phenotypic maturation of DCs Immature DCs were generated from bone marrow precursors by treatment with 2 ng/ml of GM-CSF. On day 8 of culture, non-adherent and loosely adherent cells, i.e., immature DCs, were harvested from the
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cultures. An analysis of cell-surface markers showed that more than 85% of cells were CD11c+, but no CD3+ or B220+ cells were observed. Immature DCs were treated with LPS for 24 h to induce maturation. In all experimental conditions, glabridin produced no cytotoxicity (data not shown). Glabridin at concentrations from 5–20 μM was not toxic to immature and LPS-treated mature DCs, as demonstrated by PI staining. In the following experiments, we focused on the effect of non-toxic concentrations of glabridin on the process of LPS-induced DC maturation. To investigate whether glabridin affected DC maturation, we treated immature DCs with LPS and glabridin for 24 h (Fig. 1) and 48 h (data not shown). Although the expression levels of surface molecules in immature DCs were relatively low, stimulation of cells with LPS resulted in the up-regulation of CD80 (Fig. 1A), CD86, CD40 (1B), MHC-I, and MHC-II expression (1C), which was decreased by glabridin in a dose-dependent manner. In order to rule out the competitive binding of glabridin and LPS to TLR4, we pre-treated DCs with glabridin for 24 h, washed them out three times, and then
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activated DCs with LPS for 24 h. As shown in Fig. 1D, pre-treated glabridin could inhibit LPS-induced DC maturation. To investigate whether glabridin selectively inhibited DC maturation induced by the TLR4 ligand LPS, we examined the effect of glabridin on DC maturation induced by the TLR3 ligand poly (I:C) and the TLR2 ligand zymosan. Glabridin inhibited the expression of CD86 surface marker in DCs matured with poly (I:C) (Fig. 2A) or zymosan (Fig. 2B), although it was weaker than the inhibitory potential of glabridin on LPS-induced CD86 expression. These results demonstrated that glabridin decreased the phenotypic maturation of DCs induced by various TLR ligands including LPS, poly (I:C), and zymosan, although there was some difference in inhibitory effects. 3.2. Glabridin reduces the cytokine secretion by DCs Mature DCs can secrete IL-12 and other inflammatory cytokines. To examine the effect of glabridin on cytokine production by DCs, we
Fig. 1. Glabridin inhibits LPS-induced phenotypic maturation of DCs. Immature DCs (iDCs) were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days. They were further incubated with 1 μg/ml of LPS in the presence or absence of glabridin for 24 h. Control immature DCs (UN) were not treated with LPS or glabridin. Non-adherent and loosely adherent cells were harvested and stained with two Abs, i.e., PE-conjugated CD11c Ab plus FITC-conjugated Abs to CD80 (A), CD86/40 (B), or MHC-I/II (C). DCs were pretreated with glabridin for 24 hr, washed out, and activated with LPS for 24 h (D). Representative histograms (A) and percent of positive cells are shown (B and C). Isotype control (filled histograms) shows less than 1% of positive cells. Significances were determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
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exposed DCs to LPS in the presence or absence of glabridin for 4 h, and measured the cytokine gene expression by RT-PCR. As shown in Fig. 3, mRNA expression of IL-12p40, TNF-α, IL-1β, and IFN-α/β was markedly increased upon exposure of DCs to LPS, and decreased by glabridin treatment. Inhibitory effect of glabridin on IL-12p70 protein production was confirmed by ELISA (Fig. 3C). These results indicated that glabridin could block the production of pro-inflammatory cytokines by DCs, and suggested that glabridin suppressed the functional maturation of LPS-treated DCs. 3.3. Glabridin changes the endocytic activity of DCs Antigen capture and presentation is an important feature of DC biology. Immature DCs are highly endocytic, and this feature is lost when cells mature. To examine the effect of glabridin on endocytosis of DCs, we used the fluorescent marker dextran-FITC, which is mainly taken up via the mannose receptor. Although immature DCs showed a relatively high uptake of dextran-FITC, LPS-treated mature DCs showed a decreased uptake, which was reversed by glabridin in a dose-dependent manner (Fig. 4A and B). This result demonstrated that glabridin inhibited LPS-induced DC maturation, which resulted in an increased antigen uptake. Parallel experiments were performed at 4 °C to examine nonspecific binding/uptake of dextran-FITC to DCs. These results suggested again that glabridin inhibited the functional maturation of DCs. Fig. 2. Glabridin inhibits poly (I:C)- or zymosan-induced phenotypic maturation of DCs. Immature DCs (iDCs) were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were further treated with 50 μg/ml of poly (I:C) (Pol, A) or 5 μg/ ml of zymosan (Zym, B) for 24 h. Control immature DCs (UN) were not treated with activators or glabridin. Non-adherent and loosely adherent cells were harvested and stained with PEconjugated CD11c Ab plus FITC-conjugated CD86 Ab. CD11c-PE was used to gate DCs. Percent of positive cells are shown and isotype control shows less than 1% of positive cells. Significances were determined using the ANOVA test versus LPS-treated groups (*pb 0.01).
3.4. Glabridin inhibited DC migration to chemokines Mature DCs highly express chemokine receptors, such as CXCR4 and CCR7, and migrate better than immature DCs. To examine the effect of glabridin on DC migration, we exposed DCs to LPS in the presence or absence of glabridin for 24 h, and measured the cytokine gene expression by RT-PCR. As shown in Fig. 5A, mRNA expression of
Fig. 3. Glabridin inhibits cytokine expression by DCs. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 4 h. Control immature DCs (UN) were not treated with LPS or glabridin. The mRNA levels of IL-12p40, TNF-α, IL-1β, and IFN-α/β were measured by RT-PCR (A) and band areas were analyzed using an ImageJ image analysis system and data were presented as ratios versus β-actin (B). Protein production of IL-12p70 (heterodimer of IL-12p35 and p40) was measured by ELISA (C). Significance was determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
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Fig. 4. Effect of glabridin on endocytosis of DCs. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 24 h. Control immature DCs (UN) were not treated with LPS or glabridin. Non-adherent and loosely adherent cells were harvested and treated with 0.7 mg/ml of FITC-dextran for 1 h at 37 °C (A). After washing, DCs were stained with PE-conjugated anti-CD11c Ab, and double-stained DCs were analyzed by flow cytometry. Parallel experiments were performed at 4 °C to examine nonspecific binding of dextran-FITC. Representative histograms (A) and mean (B) of three separate experiments are shown. Significances were determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
CXCR4 and CCR7 was markedly increased upon exposure of DCs to LPS, and decreased by glabridin treatment. As expected, glabridin inhibited DC migration against CXCR4 ligand SDF-1α (Fig. 5B) and CCR7 ligand MIP-3β (Fig. 5B). These results suggested that glabridin might suppress DC homing to regional lymph nodes, which was crucially dependent on MIP-3β.
3.5. Glabridin inhibits DC-mediated activation of allogenic T cells Compared with immature DCs, mature DCs strongly activate allogenic T cells to proliferate and to produce cytokines. To examine the effect of glabridin on DC-mediated T cell activation, we induced mixed lymphocyte response using C57BL/6 mouse-derived DCs (H-2b)
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Fig. 5. Glabridin inhibits DC migration against chemokines. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 24 h. Control immature DCs (UN) were not treated with LPS or glabridin. The mRNA levels of CXCR4 and CCR7 were measured by RT-PCR (A). Chemotaxis assays were performed by using a transwell chamber. DCs were added to the upper wells of transwell plate. Lower wells contained various doses of chemokines (SDF-1α or MIP-3β). The number of cells that migrated to the lower well following a 2 h incubation was counted by using a flow cytometer. Significance was determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
and BALB/c mouse-derived T cells (H-2d). As shown in Fig. 6A, T cells incubated with LPS-untreated immature DCs showed weak proliferation that was not affected by glabridin. However, T cells incubated with LPS-treated mature DCs exhibited enhanced proliferation that was decreased by glabridin. Immature DCs could not activate allo-T cells to produce IFN-γ (Fig. 6B) and IL-4 (Fig. 6C). In contrast, LPS-treated mature DCs could activate T cells to produce these cytokines, which were significantly decreased by glabridin. These results suggested that glabridin-treated DCs had a weak capacity to activate allogenic T cells to proliferate and to secrete cytokines.
3.7. Glabridin inhibits zymosan-induced inflammation in mice
3.6. Glabridin inhibits NF-κB and MAPK signaling pathways in LPS-treated DCs
4. Discussion
LPS could induce DC maturation through signaling pathways downstream of TLR4, such as MAPK and NF-κB. To investigate the effect of glabridin on DC maturation at the molecular level, we performed western blotting to examine the phosphorylation of MAPKs and the nuclear translocation of NF-κB. Basal levels of phosphorylated p38, ERK, and JNK MAPKs in immature DCs were very low (Fig. 7A). However, upon exposure to LPS, we observed a profound increase in phosphorylation of MAPKs over the basal levels in mature DCs. Glabridin dose-dependently decreased the LPSinduced phosphorylation of p38, ERK, and JNK, indicating that MAPK pathways might be involved in glabridin action on DC maturation (Fig. 7A). The majority of NF-κB subunits are sequestered in the cytoplasm by IκBα/β and translocated into the nucleus after IκBα/β degradation. As shown in Fig. 7B, total IκBα/β levels in LPStreated DCs decreased rapidly, while glabridin prevented their degradation. We also showed that glabridin blocked the nuclear translocation of NF-κB p50 and p65 (Fig. 7C). These results suggested that glabridin could inhibit LPS-induced maturation by blocking MAPK and NF-κB signaling.
When subcutaneously injected into the footpad of mice, zymosan activates monocytes including DCs and macrophages, followed by an increase of DC homing into regional popliteal lymph nodes where DCs exert their potent APC function [20]. As shown in Fig. 8, zymosan increased popliteal lymph node weights, and it was dose-dependently decreased by glabridin treatment. These results indicated that glabridin inhibited DC homing to regional lymph nodes, which might be due to the decreased chemokine receptor expression and migration to chemokines.
It has been reported that licorice is one of the most common plants prescribed by practitioners of traditional Chinese medicine and licorice-derived compounds—e.g., glycyrrhizin, isoliquiritigenin, licochalcone, and glabridin—have a variety of pharmaceutical effects [21– 23]. Among them, glabridin, a polyphenolic flavonoid and major component of the hydrophobic fraction of licorice, has been reported to have an inhibitory effect on inflammation [24,25]. Macrophages were the only inflammatory cells studied as target cells of glabridin. Glabridin inhibited inducible nitric oxide synthase (iNOS) expression and NO production in RAW 264.7 cells and peritoneal macrophages [5]. In an animal model, glabridin attenuated LPS-induced septic shock, resulting in decreased amounts of NO and TNF-α in plasma [5]. BV2 microglial cells, specialized macrophages in the brain, were also reported to be target cells of glabridin. Glabridin inhibited LPSinduced production of inflammatory mediators including NO, TNF-α, and IL-1β in BV2 cells [26]. In this study, we newly suggested DCs as inflammatory target cells of glabridin. We found that glabridin inhibited the phenotypic and functional maturation of DCs. During DC differentiation from precursors, two major stages can be identified: an immature stage
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Fig. 6. Glabridin inhibits DC-mediated alloactivation of T cells. iDCs were generated from the BM cells of C57BL/6 mice treated with 2 ng/ml of GM-CSF for 8 days. iDCs were further incubated for 24 h with glabridin only, LPS only, or LPS plus glabridin (10 μM). Control immature DCs (UN) were not treated with LPS or glabridin. Then, non-adherent and loosely adherent cells were harvested and treated with 40 μg/ml mitomycin C (MMC) for 1 h. Various numbers of MMC-treated DCs were added to 100,000 T cells purified from BALB/c mice. After 5 days of culture, T cells were labeled with [3H]thymidine and harvested using an automated cell harvester (A). Culture medium was collected 24 h after mixing DCs and T cells, and IFN-γ (B) and IL-4 (C) levels were measured by ELISA. Significance was determined using the ANOVA test versus LPStreated group (*p b 0.01).
characterized by high efficiency uptake and processing of antigens, and a mature stage characterized by the loss of antigen-uptake capacity and migration to regional lymph nodes where DCs exert their potent APC function [17]. Our data showed that glabridin prevented LPS-induced up-regulation of CD40, CD80, CD86, MHC-I and MHC-II surface molecules, whereas the endocytotic capacity of glabridintreated DC was profoundly increased. Fewer CD40 molecules on the DCs could result in diminished antigen presentation, reduced cytokine production and subsequent lack of CD80 and CD86 up-regulation [27]. Furthermore, glabridin inhibited the production of pro-inflammatory cytokines, including IL-12p40, TNF-α, and IL-1β, by LPS-stimulated DCs. Mature DC's main function is to activate allogenic T cells to proliferate and to produce cytokines, which were significantly decreased by glabridin. Overall inhibitory effect of glabridin on DC maturation was likely mild, since glabridin up to 20 μM could not completely inhibit phenotypic and functional maturation of DCs. These results suggested that glabridin partially inhibited DC maturation, resulting in partial inhibition of DC to activate allogenic T cells to proliferate and to secrete cytokines. We also investigated how glabridin inhibited DC maturation induced by TLR ligands. Glabridin could inhibit LPS-, poly (I:C)-, and zymosaninduced maturation of DCs, demonstrating that glabridin inhibited DC maturation by blocking common intracellular signaling pathways from TLR2 for zymosan, TLR4 for LPS, and TLR3 for poly (I:C). Although the detailed events downstream of each TLR differ, the transcription factor
Fig. 7. Glabridin inhibits phosphorylation of MAPKs and nuclear translocation of NF-κB p50/65. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days, and then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 15 min. Control immature DCs (UN) were not treated with LPS or glabridin. Non-phosphorylated and phosphorylated-ERK, -JNK, and -p38 were detected by immunoblotting using specific antibodies (A). iDCs were treated with 1 μg/ml LPS in the presence or absence of glabridin for 15–120 min, and total cell extracts were blotted with anti-IκBα and IκBβ antibodies (B). iDCs were incubated with LPS (1 μg/ml) or glabridin (10 μM) for 15 min. Nuclear extracts were isolated and blotted with anti-p50 and p-65 antibody (C). These results are representative of three independent experiments.
NF-κB and MAPKs are usually activated [28]. The nuclear translocation and activity of NF-κB is controlled by a family of cytoplasmic inhibitory proteins, the IκBs. The IκBs interact with NF-κB dimers, thereby blocking their nuclear translocation. Ligation of TLRs leads to phosphorylation of IκBs followed by their proteolytic degradation and the release of active NF-κB, which is then translocated in the nucleus. NF-κB controls the expression of pro-inflammatory mediators including cytokines, adhesion molecules, and co-stimulatory molecules such as MHC-I, and -II, which are expressed in DCs and markedly up-regulated when DCs change from the immature antigen-uptake mode to the mature antigen-
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References
Fig. 8. Glabridin inhibits zymosan-induced inflammation in mice. Inflammation was induced by subcutaneous injection of zymosan A (Zym, 150 μg/mouse). Naïve mice (UN) were not treated with zymosan or glabridin. Vehicle (5% Tween 80 in PBS) or glabridin was treated daily by intraperitoneal administration. On day 5, popliteal lymph nodes were isolated and weighed. Data were expressed as the mean ± STD (n = 4, A). A representative photograph is shown (B). Significance was determined using the ANOVA test versus LPS-treated group (*p b 0.01).
presenting mode [29]. In this study, we showed that glabridin inhibited the activation of NF-κB by blocking the degradation of IκBα/β and the nuclear translocation of p65/p50 in LPS-treated DCs. Consistent with this study, glabridin inhibited NF-κB activation in RAW 264.7 macrophages and BV2 microglial cells [5,26,30]. MAPK signaling pathways also play a major role in DC maturation [29,31,32]. Three MAPKs, p38, ERK, and JNK, are also involved in LPS-induced maturation of bone marrowderived DCs [33,34]. We also showed that glabridin also inhibited the phosphorylation of ERK, JNK and p38 MAPKs in LPS-treated DCs. Overall this data suggest that glabridin inhibited TLR-related signalings, including NF-κB and MAPKs in DCs. Finally, we showed that glabridin was active in an in vivo condition by using a zymosan-induced inflammation model in mice. Zymosan has been a useful tool in the study of innate immune response and monocytes, including DCs and macrophages, have been implicated in the cellular response to zymosan [20]. When injected into the footpad of mice, zymosan increased regional lymph node weights, which was due to the enhanced migration of monocytes, mainly DCs. We showed that glabridin inhibited a zymosan-induced increase of lymph node weight. It might result from the decrease of CCR7 expression and migration to MIP-3β, followed by impaired homing to lymph nodes. In summary, we demonstrated here that DCs together with macrophages might be cellular targets of glabridin and suggested that this compound inhibited the maturation and functions of DCs by blocking the activation of NF-κB and MAPKs. However, we could not clarify molecular targets of glabridin yet, which requires the continuous effects to identify target proteins interacting with glabridin in DCs. Acknowledgments This work was supported by the Korea Research Foundation Grant (MRC, R13-2008-001-00000-00) and the Korea Health 21 R&D Project (A062254).
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Inhibition of bone marrow-derived dendritic cell maturation by glabridin Jee Youn Kim a,1, Jong Soon Kang b,1, Hwan Mook Kim b, Hwa Sun Ryu a, Hyung Sook Kim a, Hong Kyung Lee a, Yeon Jin Kim a, Jin Tae Hong a, Youngsoo Kim a, Sang-Bae Han a,⁎ a b
College of Pharmacy and Medical Research Center (CICT), Chungbuk National University, 410 Seongbong, Heungduk, Cheongju, Chungbuk 361-763, Republic of Korea Korea Research Institute of Bioscience and Biotechnology, Ochang, Chungbuk 363-883, Republic of Korea
a r t i c l e
i n f o
Article history: Received 21 January 2010 Received in revised form 23 June 2010 Accepted 24 June 2010 Keywords: Glabridin Dendritic cells Maturation NF-κB MAPKs
a b s t r a c t Glabridin has multiple pharmacological activities including anti-microbial, anti-atherosclerotic, anti-nephritic, anti-inflammatory and cardiovascular protective activities. In this study, we investigated the effect of glabridin on dendritic cells, which play an essential role in innate and adaptive immune responses. Glabridin inhibited lipopolysaccharide-, poly (I:C)-, or zymosan-induced phenotypic maturation of dendritic cells (DCs), as proven by the decreased expression of CD40, CD80, CD86, MHC-I, and MHC-II. Glabridin decreased the functional maturation of DCs, in that glabridin attenuated pro-inflammatory cytokine production of IL-12, IL-1β, TNF-α, and IFN-α/β, enhanced antigen capture capacity, inhibited migration to SDF-1α and MIP-3β, and impaired induction of allogenic T cell activation. We also showed that glabridin inhibited zymosan-induced inflammation in mice. As a mode of action, we showed that glabridin inhibited degradation of IκΒα/β, nuclear translocation of NF-κB p65/ p50, and phosphorylation of ERK, JNK, and p38 MAPKs. Taken together, the present results show that glabridin inhibits dendritic cell maturation by blocking NF-κB and MAPK signalings. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Dendritic cells (DCs) are potent antigen-presenting cells that play a pivotal role in the innate and adaptive immune responses [1]. DCs originate from bone marrow precursors and migrate to almost every tissue, where they reside in an immature state [2]. Immature DCs are equipped to capture antigens and to produce large numbers of immunogenic MHC–peptide complexes. In the presence of maturation-inducing stimuli, such as inflammatory cytokines, or stimulation via CD40 ligand or toll-like receptor ligands (TLRs), including lipopolysaccharide (LPS), DCs undergo maturation with up-regulation of adhesion and co-stimulatory molecules. Mature DCs migrate to secondary lymphoid tissues and present antigen to naïve T cells and initiate T cell responses [3]. Since DCs play a key role in the initiation of the immune response, they are considered promising tools and targets for immunotherapy in several immune diseases [4]. The root of Glycyrrhiza glabra (licorice) has long been used as an antidote, demulcent, expectorant, and remedy for allergic inflammation [5]. Licorice contains glycyrrhizin, oleane triterpenoids, glucose and several flavonoids [6]. As a polyphenolic flavonoid and a major component of the hydrophobic fraction of licorice extract, glabridin, (R)-4-(3,4-dihydro-8,8-dimethyl-2H,8H-benzo[1,2-b:3,4-b′]dipyran3yl)-1,3-benzenediol, is known to possess multiple pharmacological activities. Glabridin was initially identified as an anti-microbial agent, ⁎ Corresponding author. Tel./fax: +82 43 261 2815. E-mail address: [email protected] (S.-B. Han). 1 These authors contributed equally to this work. 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.06.025
and later as an anti-melanogenic, anti-nephritic, cardiovascular protective, anti-obesity, anti-ulcer, and anti-fungal agent [6–11]. In addition, glabridin was reported to have anti-atherosclerotic activity through its inhibition of low-density lipoprotein oxidation [12]. Glabridin enhanced the efficacy of cancer chemotherapy by blocking P-glycoprotein and multidrug resistant protein 1 [13]. Interestingly, glabridin exhibited estrogen receptor agonism and growth inhibitory action in breast cancer cells [14]. Glabridin had a neuroprotective effect via modulation of neuronal cell apoptosis [15]. Among the multiple pharmacological activities of glabridin, we focused on its anti-inflammatory activity. Until now, macrophages were the only target cells of glabridin with regard to its antiinflammatory activity [5]. In this study, we investigated the suppressive activity of glabridin on other target immune cells, especially DCs, which play an important role in innate and adaptive immunity. We demonstrated that glabridin inhibited DC maturation by blocking NF-κB and MAPK signaling pathways. 2. Materials and methods 2.1. Materials Glabridin was purchased from Wako Pure Chemicals (97% purity, catalogue number 070-04821, Osaka, Japan). It was dissolved in dimethyl sulfoxide and stored at −20 °C. Female C57BL/6 (H-2b) and BALB/c (H-2d) mice (6–8 weeks old) were obtained from Korea Research Institute of Bioscience and Biotechnology (Chungbuk, Korea). Mice were housed in specific pathogen-free conditions at 21–
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24 °C and 40–60% relative humidity under a 12-h light/dark cycle. All animals were acclimatized for at least 1 week prior to the experiments. The experimental procedures used in this study were approved by the CBNU Animal Experimentation Ethics Committee. Anti-mouse antibodies against CD11c, CD40, CD80, CD86, MHC-I and MHC-II were purchased from BD Biosciences (San Jose, CA, USA) and ERK, p38, and JNK were purchased from Cell Signaling (Danvers, MA, USA). 2.2. Generation of bone marrow-derived DCs DCs were generated from bone marrow (BM) cells obtained from 6–7-week-old female mice [16]. Briefly, BM cells were flushed out from femurs and tibias. After red blood cells were lysed, whole BM cells (2 × 105 cells/ml) were cultured in 100-mm2 culture dishes in 10 ml/dish of complete medium containing 2 ng/ml GM-CSF (R&D Systems, Minneapolis, MN, USA). On day 3, another 10 ml of fresh complete medium containing 2 ng/ml GM-CSF was added, and half of the medium was changed on day 6. On day 8, non-adherent and loosely adherent DCs were harvested by vigorous pipetting and used as immature DCs (iDCs). iDCs recovered from these cultures were generally N85% CD11c+, but not CD3+ and B220+. 2.3. Phenotype analysis Phenotypic maturation of DCs was analyzed by flow cytometry [17]. Cell staining was performed using a combination of FITCconjugated anti-CD40, anti-CD80, anti-CD86, or anti-MHC plus PEconjugated CD11c antibodies. Cells were analyzed using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using WinMdi software (Scripps, La Jolla, CA, USA). Forward and side scatter parameters were used to gate live cells. Cell viability was examined by the propidium iodide (PI) nuclear staining method. Cells were stained with 1 μg/ml of PI and analyzed with a FACSCanto flow cytometer. Cells stained with PI were considered dead cells [18].
G-3′, antisense, 5′-TGA CGT CAT AGG CAA TGT TGA GCT-3′; and β-actin, sense, 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′, antisense 5′-TAA AAC GCA GCT CAG TAACAG TCC G-3′. PCR products were fractionated on 1% agarose gels and stained with 5 μg/ml ethidium bromide [17]. 2.7. Western blots Cell lysates were prepared as previously described [17]. Detergentinsoluble materials were removed, and equal amounts of protein were fractionated by 10% SDS-PAGE and transferred to pure nitrocellulose membranes. Membranes were blocked with 5% BSA in Tween 20 plus TBS (TTBS) for 1 h and then incubated with an appropriate dilution of primary antibody in 5% BSA (in TTBS) for 2 h. Blots were incubated with biotinylated antibody for 1 h and further incubated with streptavidin conjugated to HRP for 1 h. Signals were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.8. Mixed lymphocyte reaction (MLR) Responder T cells were purified from the spleen of BALB/c mice by negative depletion using biotinylated antibodies for B220, GR-1, and CD11c (BD Pharmingen, San Diego, CA, USA) and Dynabeads M-280 streptavidin (Invitrogen, Dynal, Carlsbad, CA, USA), as previously described [19]. Purity was typically more than 90%. DCs generated from the BM cells of C57BL/6 mice were treated with 40 μg/ml mitomycin C (MMC) for 1 h. One million MMC-treated DCs were added to 1 × 105 T cells in U-bottom 96-well plates (activator:responder cell ratio = 10). Allogenic T cells were pulsed with [3H]-thymidine (113 Ci/nmol, NEN, Boston, MA, USA) at a concentration of 1 μCi/well for the last 18 h and then harvested on day 5 using an automated cell harvester (Innotech, Dottikon, Switzerland). The amount of [3H]thymidine incorporated into the cells was measured using a Wallac Microbeta scintillation counter (Wallac, Turku, Finland) [16]. 2.9. Zymosan-induced inflammation
2.4. Endocytosis assay To analyze the endocytosis of DCs, 4 × 105 DCs were incubated at 37 °C for 1 h with 0.7 mg/ml FITC-dextran (42,000 Da, Sigma-Aldrich, St. Louis, MO, USA). After incubation, cells were washed twice with cold washing buffer (PBS containing 0.5% BSA) and stained using PEconjugated anti-CD11c antibody. Double-stained DCs were analyzed by flow cytometry. In addition, parallel experiments were performed at 4 °C to determine the nonspecific binding of FITC-dextran to DCs [17].
Inflammation was induced by subcutaneous injection of 50 μl of 3 mg/ ml zymosan A isolated from Saccharomyces cereviciae (Sigma-Aldrich, St. Louis, MO, USA) into the hind footpad of C57BL/6 mice. Glabridin was intraperitoneally administered from day 0 to day 5. On day 6, right and left popliteal lymph nodes (pLNs) were isolated and weighed [20]. 2.10. Chemotaxis
Levels of IL-12p70, IFN-γ and IL-4 in culture supernatants were measured using commercial immunoassay kits (R&D Systems, Minneapolis, MN, USA).
Chemotaxis assays were performed by using a transwell chamber as previously described [19]. DCs were added in a volume of 100 μl to the upper wells of a 24-well transwell plate with a 5 μm insert. Lower wells contained various doses of chemokines in 600 μl complete RPMI 1640 medium. The number of cells that migrated to the lower well following a 2 h incubation was counted by using a flow cytometer.
2.6. Reverse transcription-polymerase chain reaction (RT-PCR)
2.11. Statistics
Total RNA was isolated using TRIZOL™ Reagent (Molecular Research Center, Cincinnati, OH, USA). For RT-PCR, single-strand cDNA was synthesized from 2 μg total RNA. The primer sequences used were as follows: IL-12p40, sense, 5′-AGA GGT GGA CTG GAC TCC CGA-3′, antisense, 5′-TTT GGT GCT TCA CAC TTC AG-3′; TNF-α, sense, 5′-AGG TTC TGT CCC TTT CAC TCA CTG-3′, antisense, 5′-AGA GAA CCT GGG AGT CAA GGT A-3′; IL-1β, sense, 5′-ATG GCA ATG TTC CTG AAC TCA ACT-3′, antisense, 5′-CAG GAC AGG TAT AGA TTC TTT CCT TT-3′; IFN-α, sense, 5′TCT GAT GCA GCA GGT GGG-3′, antisense, 5′-AGG GCT CTC CAG AYT TCT GCT CTG-3′; IFN-β, sense, 5′-CCA CAG CCC TCT CCA TCA ACT ATA AGC-3′, antisense, 5′-AGC TCT TCA ACT GGA GAG CAG TTG AGG-3′; CXCR4, sense, 5′-GGC TGT AGA GCG AGT GTT GC-3′, antisense, 5′-GTA GAG GTT GAC AGT GTA GAT-3′; CCR7, sense, 5′-ACA GCG GCC TCC AGA AGA ACA GCG
Data represent the mean +/− STD of more than three samples and all experiments were performed more than three times. Standard deviations (STD) were calculated using the Student's t test and p values were calculated using ANOVA software (GraphPad Software, San Diego, CA, USA).
2.5. Measurement of secreted IL-12p70, IFN-γ and IL-4
3. Results 3.1. Glabridin blocks the phenotypic maturation of DCs Immature DCs were generated from bone marrow precursors by treatment with 2 ng/ml of GM-CSF. On day 8 of culture, non-adherent and loosely adherent cells, i.e., immature DCs, were harvested from the
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cultures. An analysis of cell-surface markers showed that more than 85% of cells were CD11c+, but no CD3+ or B220+ cells were observed. Immature DCs were treated with LPS for 24 h to induce maturation. In all experimental conditions, glabridin produced no cytotoxicity (data not shown). Glabridin at concentrations from 5–20 μM was not toxic to immature and LPS-treated mature DCs, as demonstrated by PI staining. In the following experiments, we focused on the effect of non-toxic concentrations of glabridin on the process of LPS-induced DC maturation. To investigate whether glabridin affected DC maturation, we treated immature DCs with LPS and glabridin for 24 h (Fig. 1) and 48 h (data not shown). Although the expression levels of surface molecules in immature DCs were relatively low, stimulation of cells with LPS resulted in the up-regulation of CD80 (Fig. 1A), CD86, CD40 (1B), MHC-I, and MHC-II expression (1C), which was decreased by glabridin in a dose-dependent manner. In order to rule out the competitive binding of glabridin and LPS to TLR4, we pre-treated DCs with glabridin for 24 h, washed them out three times, and then
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activated DCs with LPS for 24 h. As shown in Fig. 1D, pre-treated glabridin could inhibit LPS-induced DC maturation. To investigate whether glabridin selectively inhibited DC maturation induced by the TLR4 ligand LPS, we examined the effect of glabridin on DC maturation induced by the TLR3 ligand poly (I:C) and the TLR2 ligand zymosan. Glabridin inhibited the expression of CD86 surface marker in DCs matured with poly (I:C) (Fig. 2A) or zymosan (Fig. 2B), although it was weaker than the inhibitory potential of glabridin on LPS-induced CD86 expression. These results demonstrated that glabridin decreased the phenotypic maturation of DCs induced by various TLR ligands including LPS, poly (I:C), and zymosan, although there was some difference in inhibitory effects. 3.2. Glabridin reduces the cytokine secretion by DCs Mature DCs can secrete IL-12 and other inflammatory cytokines. To examine the effect of glabridin on cytokine production by DCs, we
Fig. 1. Glabridin inhibits LPS-induced phenotypic maturation of DCs. Immature DCs (iDCs) were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days. They were further incubated with 1 μg/ml of LPS in the presence or absence of glabridin for 24 h. Control immature DCs (UN) were not treated with LPS or glabridin. Non-adherent and loosely adherent cells were harvested and stained with two Abs, i.e., PE-conjugated CD11c Ab plus FITC-conjugated Abs to CD80 (A), CD86/40 (B), or MHC-I/II (C). DCs were pretreated with glabridin for 24 hr, washed out, and activated with LPS for 24 h (D). Representative histograms (A) and percent of positive cells are shown (B and C). Isotype control (filled histograms) shows less than 1% of positive cells. Significances were determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
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exposed DCs to LPS in the presence or absence of glabridin for 4 h, and measured the cytokine gene expression by RT-PCR. As shown in Fig. 3, mRNA expression of IL-12p40, TNF-α, IL-1β, and IFN-α/β was markedly increased upon exposure of DCs to LPS, and decreased by glabridin treatment. Inhibitory effect of glabridin on IL-12p70 protein production was confirmed by ELISA (Fig. 3C). These results indicated that glabridin could block the production of pro-inflammatory cytokines by DCs, and suggested that glabridin suppressed the functional maturation of LPS-treated DCs. 3.3. Glabridin changes the endocytic activity of DCs Antigen capture and presentation is an important feature of DC biology. Immature DCs are highly endocytic, and this feature is lost when cells mature. To examine the effect of glabridin on endocytosis of DCs, we used the fluorescent marker dextran-FITC, which is mainly taken up via the mannose receptor. Although immature DCs showed a relatively high uptake of dextran-FITC, LPS-treated mature DCs showed a decreased uptake, which was reversed by glabridin in a dose-dependent manner (Fig. 4A and B). This result demonstrated that glabridin inhibited LPS-induced DC maturation, which resulted in an increased antigen uptake. Parallel experiments were performed at 4 °C to examine nonspecific binding/uptake of dextran-FITC to DCs. These results suggested again that glabridin inhibited the functional maturation of DCs. Fig. 2. Glabridin inhibits poly (I:C)- or zymosan-induced phenotypic maturation of DCs. Immature DCs (iDCs) were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were further treated with 50 μg/ml of poly (I:C) (Pol, A) or 5 μg/ ml of zymosan (Zym, B) for 24 h. Control immature DCs (UN) were not treated with activators or glabridin. Non-adherent and loosely adherent cells were harvested and stained with PEconjugated CD11c Ab plus FITC-conjugated CD86 Ab. CD11c-PE was used to gate DCs. Percent of positive cells are shown and isotype control shows less than 1% of positive cells. Significances were determined using the ANOVA test versus LPS-treated groups (*pb 0.01).
3.4. Glabridin inhibited DC migration to chemokines Mature DCs highly express chemokine receptors, such as CXCR4 and CCR7, and migrate better than immature DCs. To examine the effect of glabridin on DC migration, we exposed DCs to LPS in the presence or absence of glabridin for 24 h, and measured the cytokine gene expression by RT-PCR. As shown in Fig. 5A, mRNA expression of
Fig. 3. Glabridin inhibits cytokine expression by DCs. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 4 h. Control immature DCs (UN) were not treated with LPS or glabridin. The mRNA levels of IL-12p40, TNF-α, IL-1β, and IFN-α/β were measured by RT-PCR (A) and band areas were analyzed using an ImageJ image analysis system and data were presented as ratios versus β-actin (B). Protein production of IL-12p70 (heterodimer of IL-12p35 and p40) was measured by ELISA (C). Significance was determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
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Fig. 4. Effect of glabridin on endocytosis of DCs. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 24 h. Control immature DCs (UN) were not treated with LPS or glabridin. Non-adherent and loosely adherent cells were harvested and treated with 0.7 mg/ml of FITC-dextran for 1 h at 37 °C (A). After washing, DCs were stained with PE-conjugated anti-CD11c Ab, and double-stained DCs were analyzed by flow cytometry. Parallel experiments were performed at 4 °C to examine nonspecific binding of dextran-FITC. Representative histograms (A) and mean (B) of three separate experiments are shown. Significances were determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
CXCR4 and CCR7 was markedly increased upon exposure of DCs to LPS, and decreased by glabridin treatment. As expected, glabridin inhibited DC migration against CXCR4 ligand SDF-1α (Fig. 5B) and CCR7 ligand MIP-3β (Fig. 5B). These results suggested that glabridin might suppress DC homing to regional lymph nodes, which was crucially dependent on MIP-3β.
3.5. Glabridin inhibits DC-mediated activation of allogenic T cells Compared with immature DCs, mature DCs strongly activate allogenic T cells to proliferate and to produce cytokines. To examine the effect of glabridin on DC-mediated T cell activation, we induced mixed lymphocyte response using C57BL/6 mouse-derived DCs (H-2b)
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Fig. 5. Glabridin inhibits DC migration against chemokines. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days; the cells were then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 24 h. Control immature DCs (UN) were not treated with LPS or glabridin. The mRNA levels of CXCR4 and CCR7 were measured by RT-PCR (A). Chemotaxis assays were performed by using a transwell chamber. DCs were added to the upper wells of transwell plate. Lower wells contained various doses of chemokines (SDF-1α or MIP-3β). The number of cells that migrated to the lower well following a 2 h incubation was counted by using a flow cytometer. Significance was determined using the ANOVA test versus LPS-treated groups (*p b 0.01).
and BALB/c mouse-derived T cells (H-2d). As shown in Fig. 6A, T cells incubated with LPS-untreated immature DCs showed weak proliferation that was not affected by glabridin. However, T cells incubated with LPS-treated mature DCs exhibited enhanced proliferation that was decreased by glabridin. Immature DCs could not activate allo-T cells to produce IFN-γ (Fig. 6B) and IL-4 (Fig. 6C). In contrast, LPS-treated mature DCs could activate T cells to produce these cytokines, which were significantly decreased by glabridin. These results suggested that glabridin-treated DCs had a weak capacity to activate allogenic T cells to proliferate and to secrete cytokines.
3.7. Glabridin inhibits zymosan-induced inflammation in mice
3.6. Glabridin inhibits NF-κB and MAPK signaling pathways in LPS-treated DCs
4. Discussion
LPS could induce DC maturation through signaling pathways downstream of TLR4, such as MAPK and NF-κB. To investigate the effect of glabridin on DC maturation at the molecular level, we performed western blotting to examine the phosphorylation of MAPKs and the nuclear translocation of NF-κB. Basal levels of phosphorylated p38, ERK, and JNK MAPKs in immature DCs were very low (Fig. 7A). However, upon exposure to LPS, we observed a profound increase in phosphorylation of MAPKs over the basal levels in mature DCs. Glabridin dose-dependently decreased the LPSinduced phosphorylation of p38, ERK, and JNK, indicating that MAPK pathways might be involved in glabridin action on DC maturation (Fig. 7A). The majority of NF-κB subunits are sequestered in the cytoplasm by IκBα/β and translocated into the nucleus after IκBα/β degradation. As shown in Fig. 7B, total IκBα/β levels in LPStreated DCs decreased rapidly, while glabridin prevented their degradation. We also showed that glabridin blocked the nuclear translocation of NF-κB p50 and p65 (Fig. 7C). These results suggested that glabridin could inhibit LPS-induced maturation by blocking MAPK and NF-κB signaling.
When subcutaneously injected into the footpad of mice, zymosan activates monocytes including DCs and macrophages, followed by an increase of DC homing into regional popliteal lymph nodes where DCs exert their potent APC function [20]. As shown in Fig. 8, zymosan increased popliteal lymph node weights, and it was dose-dependently decreased by glabridin treatment. These results indicated that glabridin inhibited DC homing to regional lymph nodes, which might be due to the decreased chemokine receptor expression and migration to chemokines.
It has been reported that licorice is one of the most common plants prescribed by practitioners of traditional Chinese medicine and licorice-derived compounds—e.g., glycyrrhizin, isoliquiritigenin, licochalcone, and glabridin—have a variety of pharmaceutical effects [21– 23]. Among them, glabridin, a polyphenolic flavonoid and major component of the hydrophobic fraction of licorice, has been reported to have an inhibitory effect on inflammation [24,25]. Macrophages were the only inflammatory cells studied as target cells of glabridin. Glabridin inhibited inducible nitric oxide synthase (iNOS) expression and NO production in RAW 264.7 cells and peritoneal macrophages [5]. In an animal model, glabridin attenuated LPS-induced septic shock, resulting in decreased amounts of NO and TNF-α in plasma [5]. BV2 microglial cells, specialized macrophages in the brain, were also reported to be target cells of glabridin. Glabridin inhibited LPSinduced production of inflammatory mediators including NO, TNF-α, and IL-1β in BV2 cells [26]. In this study, we newly suggested DCs as inflammatory target cells of glabridin. We found that glabridin inhibited the phenotypic and functional maturation of DCs. During DC differentiation from precursors, two major stages can be identified: an immature stage
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Fig. 6. Glabridin inhibits DC-mediated alloactivation of T cells. iDCs were generated from the BM cells of C57BL/6 mice treated with 2 ng/ml of GM-CSF for 8 days. iDCs were further incubated for 24 h with glabridin only, LPS only, or LPS plus glabridin (10 μM). Control immature DCs (UN) were not treated with LPS or glabridin. Then, non-adherent and loosely adherent cells were harvested and treated with 40 μg/ml mitomycin C (MMC) for 1 h. Various numbers of MMC-treated DCs were added to 100,000 T cells purified from BALB/c mice. After 5 days of culture, T cells were labeled with [3H]thymidine and harvested using an automated cell harvester (A). Culture medium was collected 24 h after mixing DCs and T cells, and IFN-γ (B) and IL-4 (C) levels were measured by ELISA. Significance was determined using the ANOVA test versus LPStreated group (*p b 0.01).
characterized by high efficiency uptake and processing of antigens, and a mature stage characterized by the loss of antigen-uptake capacity and migration to regional lymph nodes where DCs exert their potent APC function [17]. Our data showed that glabridin prevented LPS-induced up-regulation of CD40, CD80, CD86, MHC-I and MHC-II surface molecules, whereas the endocytotic capacity of glabridintreated DC was profoundly increased. Fewer CD40 molecules on the DCs could result in diminished antigen presentation, reduced cytokine production and subsequent lack of CD80 and CD86 up-regulation [27]. Furthermore, glabridin inhibited the production of pro-inflammatory cytokines, including IL-12p40, TNF-α, and IL-1β, by LPS-stimulated DCs. Mature DC's main function is to activate allogenic T cells to proliferate and to produce cytokines, which were significantly decreased by glabridin. Overall inhibitory effect of glabridin on DC maturation was likely mild, since glabridin up to 20 μM could not completely inhibit phenotypic and functional maturation of DCs. These results suggested that glabridin partially inhibited DC maturation, resulting in partial inhibition of DC to activate allogenic T cells to proliferate and to secrete cytokines. We also investigated how glabridin inhibited DC maturation induced by TLR ligands. Glabridin could inhibit LPS-, poly (I:C)-, and zymosaninduced maturation of DCs, demonstrating that glabridin inhibited DC maturation by blocking common intracellular signaling pathways from TLR2 for zymosan, TLR4 for LPS, and TLR3 for poly (I:C). Although the detailed events downstream of each TLR differ, the transcription factor
Fig. 7. Glabridin inhibits phosphorylation of MAPKs and nuclear translocation of NF-κB p50/65. iDCs were generated from mouse BM cells by treating them with 2 ng/ml of GM-CSF for 8 days, and then further treated with 1 μg/ml LPS in the presence or absence of glabridin for 15 min. Control immature DCs (UN) were not treated with LPS or glabridin. Non-phosphorylated and phosphorylated-ERK, -JNK, and -p38 were detected by immunoblotting using specific antibodies (A). iDCs were treated with 1 μg/ml LPS in the presence or absence of glabridin for 15–120 min, and total cell extracts were blotted with anti-IκBα and IκBβ antibodies (B). iDCs were incubated with LPS (1 μg/ml) or glabridin (10 μM) for 15 min. Nuclear extracts were isolated and blotted with anti-p50 and p-65 antibody (C). These results are representative of three independent experiments.
NF-κB and MAPKs are usually activated [28]. The nuclear translocation and activity of NF-κB is controlled by a family of cytoplasmic inhibitory proteins, the IκBs. The IκBs interact with NF-κB dimers, thereby blocking their nuclear translocation. Ligation of TLRs leads to phosphorylation of IκBs followed by their proteolytic degradation and the release of active NF-κB, which is then translocated in the nucleus. NF-κB controls the expression of pro-inflammatory mediators including cytokines, adhesion molecules, and co-stimulatory molecules such as MHC-I, and -II, which are expressed in DCs and markedly up-regulated when DCs change from the immature antigen-uptake mode to the mature antigen-
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References
Fig. 8. Glabridin inhibits zymosan-induced inflammation in mice. Inflammation was induced by subcutaneous injection of zymosan A (Zym, 150 μg/mouse). Naïve mice (UN) were not treated with zymosan or glabridin. Vehicle (5% Tween 80 in PBS) or glabridin was treated daily by intraperitoneal administration. On day 5, popliteal lymph nodes were isolated and weighed. Data were expressed as the mean ± STD (n = 4, A). A representative photograph is shown (B). Significance was determined using the ANOVA test versus LPS-treated group (*p b 0.01).
presenting mode [29]. In this study, we showed that glabridin inhibited the activation of NF-κB by blocking the degradation of IκBα/β and the nuclear translocation of p65/p50 in LPS-treated DCs. Consistent with this study, glabridin inhibited NF-κB activation in RAW 264.7 macrophages and BV2 microglial cells [5,26,30]. MAPK signaling pathways also play a major role in DC maturation [29,31,32]. Three MAPKs, p38, ERK, and JNK, are also involved in LPS-induced maturation of bone marrowderived DCs [33,34]. We also showed that glabridin also inhibited the phosphorylation of ERK, JNK and p38 MAPKs in LPS-treated DCs. Overall this data suggest that glabridin inhibited TLR-related signalings, including NF-κB and MAPKs in DCs. Finally, we showed that glabridin was active in an in vivo condition by using a zymosan-induced inflammation model in mice. Zymosan has been a useful tool in the study of innate immune response and monocytes, including DCs and macrophages, have been implicated in the cellular response to zymosan [20]. When injected into the footpad of mice, zymosan increased regional lymph node weights, which was due to the enhanced migration of monocytes, mainly DCs. We showed that glabridin inhibited a zymosan-induced increase of lymph node weight. It might result from the decrease of CCR7 expression and migration to MIP-3β, followed by impaired homing to lymph nodes. In summary, we demonstrated here that DCs together with macrophages might be cellular targets of glabridin and suggested that this compound inhibited the maturation and functions of DCs by blocking the activation of NF-κB and MAPKs. However, we could not clarify molecular targets of glabridin yet, which requires the continuous effects to identify target proteins interacting with glabridin in DCs. Acknowledgments This work was supported by the Korea Research Foundation Grant (MRC, R13-2008-001-00000-00) and the Korea Health 21 R&D Project (A062254).
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