Polysaccharide purified from Polyporus umbellatus (Per) Fr induces the activation and maturation of murine bone-derived dendritic cells via toll-like receptor 4

Cellular Immunology 265 (2010) 50–56

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Polysaccharide purified from Polyporus umbellatus (Per) Fr induces the activation and maturation of murine bone-derived dendritic cells via toll-like receptor 4 Xinqun Li a, Wen Xu b,*, Jun Chen a a b

First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325035, China Department of Microbiology and Immunology, Wenzhou Medical College, Wenzhou, Zhejiang 325035, China

a r t i c l e

i n f o

Article history: Received 24 March 2010 Accepted 8 July 2010 Available online 14 July 2010 Keywords: Dendritic cells Polyporus polysaccharide Toll-like receptor 4

a b s t r a c t In this study, we report that a polysaccharide isolated from a Chinese medicinal herb, Zhu Ling (the sclerotium of Polyporus umbellatus (Per) Fr), induces phenotypic and functional maturation of murine bonederived dendritic cells (BMDCs). Treatment of BMDCs with Polyporus polysaccharide (PPS) resulted in enhanced cell-surface expression of CD86, as well as enhanced production of both interleukin (IL)-12 p40 and IL-10 in a dose-dependent manner. In addition, treatment of BMDCs with PPS resulted in increased T cell-stimulatory capacity and decreased phagocytic ability. PPS-induced production of IL-12 p40 was inhibited by monoclonal antibodies to Toll-like receptor 4 (TLR4). Flow cytometric analysis showed that fluorescence-labeled PPS (f-PPS) bound specifically to BMDCs. This binding was blocked by both unlabeled PPS and anti-TLR4, but not by anti-TLR2 and anti-CR3 monoclonal antibodies. Taken together, our data show that PPS promotes the activation and maturation of murine BMDCs via TLR4. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Zhu Ling, the sclerotium of Polyporus umbellatus (Per) Fr, is a common Chinese herbal medicine widely used in China for more than 2000 years. It is traditionally used as a potent antibiotic and anti-tumor agent, a diuretic and to treat urinary tract infections. One of its bioactive components is Polyporus umbellatus polysaccharide (PPS). PPS is particularly effective in inhibiting tumor growth [1,2]. Despite numerous studies, the exact immunopharmacological properties (e.g. the immunopotentiating mechanism) of the active polysaccharide have remained largely unknown. However, we speculated that the immunostimulatory activity of PPS is mediated via the activation of dendritic cells (DCs). DCs represent a class of antigen-presenting cells (APCs) that initiate the majority of immune responses. DCs have numerous specialized features that make them extremely efficient in capturing and presenting antigens and at activating naïve T cells [3]. They are responsible for launching acquired immune responses; particularly, primary responses. Under normal conditions, tissue dendritic cells are said to be immature because they express very low levels of MHC class II and co-stimulatory molecules on their surfaces, and are unable to activate T cells [4,5]. In peripheral tissues, these cells capture and process antigen and migrate to

* Corresponding author. E-mail address: [email protected] (W. Xu). 0008-8749/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.07.002

secondary lymphoid organs where they mature. Once in the T cell-rich zones of the lymphoid tissues, the fully mature DCs begin to secrete substances that attract and stimulate T cells specific for the processed antigen [6]. Mature DCs also express high levels of the T cell co-stimulatory molecules CD80 and CD86, along with cell adhesion molecules such as ICAM-1 and LFA-3 [7]. Their unique efficiency at presenting antigen, and at attracting and activating specific T cells, make mature DCs the most potent APCs known. DC maturation can be induced by bacterial lipopolysaccharide (LPS) [8] or inflammatory cytokines, such as TNF [9]. Although these mediators are potent stimuli of DC maturation, they are toxic and have limited clinical applications. In this regard, nontoxic mediators that can induce DC maturation are valuable. In this study, we report that PPS induces the differentiation/maturation of murine bone marrow DCs (BMDCs). PPS-stimulated BMDC production of IL-12 p40 up-regulates the expression of co-stimulatory molecules and enhances the stimulation of naïve T cells via TLR4.

2. Materials and methods 2.1. Mice Female C3H/HeN, BALB/c and C57BL/6 mice (6–8 weeks old) were purchased from Shanghai Laboratory Animal Center, China. Animals were maintained and used in strict 66 accordance with the guidelines issued by the Beijing Government on Animal Care.

X. Li et al. / Cellular Immunology 265 (2010) 50–56

2.2. Reagents All cells were cultured in RPMI-1640 supplemented with 10% (v/v) FCS (Hyclone), penicillin/streptomycin (100 U/ml), L-glutamine (2 mM), and 2-ME (5  10 5 M). LPS from Escherichia coli, B5:55 (Sigma). Fluorescein isothiocyanate (FITC)-conjugated antiCD11c and phycoerythrin (PE)-conjugated anti-CD86 monoclonal antibodies (mAbs) were purchased from R&D Systems. Neutralizing antibodies against TLR2, TLR4 and anti-mouse CD11b (CR3) mAb were purchased from eBiosciences. Antibodies were dialyzed extensively against PBS to remove sodium azide before use in cell culture. Dextran (70 kDa) was obtained from Sigma. Mouse IL-1b, TNF-a, IL-10 and IL-12 p40 ELISA kits and were purchased from R&D Systems. The LAL assay kit was purchased from Sigma. 2.3. Preparation of PPS PPS was isolated from the boiling water extract of Zhuling, followed by ethanol precipitation, dialysis, and protein depletion using the Sevag method. The resultant polysaccharide extract was dialyzed against water and then lyophilized. The sugar content of sample was about 89.7% as measured by the phenol–sulfuric acid colorimetric method. Weight-average molecular weight (MW) was determined using size exclusion chromatography (SEC) combined with static light scattering (SLS). Wyatt Technology Astra software was used for data collection and analysis. The PPS molecules had a MW of approximately 1.6  105 and the molecular weight distribution (Mr/Mn) was 2.914. The monosaccharide of PPS was analyzed by Gas Chromatography (GC). The results showed that PPS was mainly composed of D-glucose units, but also contained D-galactose and D-mannose. The structure of PPS was determined by Fourier transform infrared spectroscopy (FTIR; Thermo Nicolet), which showed that PPS had the characteristic absorption peak of a b-D-glucopyranoside (see Supplementary materials). PPS was dissolved in RPMI-1640 medium (Gibco), filtered through a 0.22 lm filter, and stored at 20 °C for future use. 2.4. Determination of endotoxin contamination The endotoxin concentration in the PPS was measured using a limulus amebocyte lysate (LAL) chromogenic assay kit (Yihua Biotech, Shanghai, China) according to the manufacturer’s instructions. For further ruling out the possibility of LPS contamination, PPS (50 lg/ml) or LPS (1 lg/ml) was preincubated with polymyxin B (10 lg/ml) (Sigma) for 15 min at room temperature. Control samples were not incubated with polymyxin B. These preparations were then used to stimulate C3H/HeN mouse macrophages. After 20 h, culture supernatants were harvested and the levels of interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) were measured by ELISA. 2.5. Preparation of fluorescence-labeled PPS and dextran Fluorescence-labeled PPS and dextran were generated as described by Glabe et al. [10]. Briefly, PPS or dextran solution (20 mg/ ml in 1 ml water) was mixed with 0.2 ml CNBr (50 mg/ml in H2O), and maintained at pH 11 for 15 min by addition of 0.2 M NaOH. After dialysis against sodium borate buffer at pH 8.0 for 20 h, the CNBr-activated PPS or dextran was mixed with 2 mg fluoresceinamine (Sigma) for 10 h in a volume of less than 4 ml. The resultant fluorescence-labeled polysaccharide was separated from the free fluoresceinamine by gel filtration on a Sephadex G-50 (Pharmacia) column. Elution fractions (1 ml/tube) were collected and the concentration of fluoresceinamine was determined by absorbance at 440 nm. Fluorescence-

51

labeled polysaccharide fractions were mixed, adjusted to 1 mg/ml, and stored at 20 °C for future use. The concentration of fluoresceinamine in the f-APS and f-dextran preparations was approximately 5 lg/ml. 2.6. Proliferation assays Splenocytes (4  105) from C57BL/6 mice were cultured in round-bottomed 96-well plates (Nunc) in a volume of 200 ll/well in the presence of different concentrations of PPS, f-PPS or dextran (50 lg/ml). The cultures were incubated at 37 °C with 5% CO2 for 3 days. During the last 8 h of incubation, 0.2 lCi 3H-thymidine was added into each well. Thereafter, cells were harvested onto fiberglass filters and radioactivity measured using a scintillation counter (EG & G Wallac). 2.7. Generation of BMDCs BMDCs were prepared as previously described with minor modifications [11]. Briefly, bone marrow cells were flushed from the femur and tibiae of C57BL/6 mice and treated with ACK lysis buffer (Sigma) to lyze erythrocytes. Cells, at a starting number of 2  106 cells/ml, were cultured in RPMI-1640 complete media in 6-well flat bottom plates (Nunc, Denmark) at 37 °C and 5% CO2, supplemented with 10 ng/ml recombinant murine GM-CSF (R&D Systems) and recombinant murine IL-4 (R&D Systems). On day 5, non-adherent DCs were positively purified with microbead-conjugated anti-CD11c mAb by MACS columns (Miltenyi Biotec). CD11c+ BMDCs were incubated at a concentration of 1  105 cells/ml with different concentrations (12.5, 25, 50, 100 lg/ml) of PPS, serumfree RPMI-1640 or 100 ng/ml LPS. On day 7, cells and culture supernatants were collected for further experiments and analysis. 2.8. Flow cytometry BMDCs were harvested and washed with staining buffer (phosphate-buffered saline (PBS) containing 5% fetal calf serum). Cells (1–5  105) were incubated with excess anti-mouse CD16/32 to block Fc receptors and then stained with 1 lg FITC-conjugated anti-CD11c, PE-conjugated anti-CD86 or isotype-matched controls for 30 min on ice. Stained cells were then washed twice, resuspended in cold buffer and analyzed by flow cytometry (FACSAria with CellQuest software, BD Biosciences). 2.9. Cytokine ELlSA The IL-12 p40 and IL-10 levels in the BMDC culture supernatants were assayed using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. 2.10. Neutralization experiments BMDCs were preincubated for 1 h with 20 lg/ml anti-TLR2, -TLR4, -CR3 or isotype control mAbs. PPS (50 lg/ml) was then added and incubated for 24 h. The cell culture supernatants were collected and the IL-12 p40 levels measured. 2.11. Fluorescence-labeled dextran uptake The phagocytic activity of BMDCs was measured by assaying the uptake of f-dextran as previously described [12]. Briefly, day 7 BMDCs were cultured with f-dextran (1 mg/ml) at 37 °C or 4 °C for 1 h. Finally, the cells were washed three times with cold buffer and analyzed using a FACSAria as described above.

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2.12. Allogeneic mixed leukocyte reaction (MLR) CD4+CD45RA+ T cells were purified from H-2d BALB/c responder spleen lymphocytes using T cell isolation kit (Miltenyi Biotec). The allogeneic T cells obtained were distributed at 1  105 cells/well and incubated for 72 h in the presence of graded numbers of irradiated BMDCs (3000 rad). During the last 8 h of incubation, 3H-thymidine (0.2 lCi/well) was added to each well. The cells were harvested using a 96-well plate harvester (Tomtec, USA), placed onto fiberglass filters and the radioactivity on the filter mat was counted using a MicroBeta Trilux LSC counter (EG & G Wallac). 2.13. Statistical analysis Data are presented as the mean ± standard deviation (SD) of at least three individual experiments. Student’s t-test was used to analyze the results and a P-value of less than 0.05 was considered statistically significant. 3. Results 3.1. PPS-induces splenocyte activation To determine the immunostimulatory capacity of PPS, freshly separated splenocytes were tested for their ability to respond to PPS stimulation in proliferation assays. As shown in Fig. 1, PPS induced vigorous proliferation of splenocytes. In addition, f-PPS was equally effective in stimulating splenocytes, suggesting that conjugation of PPS to fluoresceinamine did not significantly alter its immunobiological activity, and so could be used in subsequent experiments. 3.2. Endotoxin testing The level of endotoxin in the PPS preparation was determined using the Limulus amoebocyte lysate (LAL) assay and was found to be 0.054 EU/ml (<0.1 EU/ml of endotoxin activity (Table 1), corresponding to 8–25 pg/ml of LPS, depending on the LPS type). In addition, as shown in Table 2, polymyxin B significantly inhibited LPS-, but not PPS-induced, production of both IL-1b and TNF-a. Therefore, the effect of PPS on macrophages was not due to endotoxin contamination. These results indicated that the level of LPS contamination in the PPS was negligible. 3.3. PPS up-regulates the co-expression of CD86 and CD11c on BMDCs

3

H-Thymidine incorporation (cpm x 1000)

To determine whether PPS can regulate the development of BMDCs in vitro, we compared the phenotypes of BMDCs treated with

Fig. 1. PPS-induced proliferation of mouse splenic cells. Splenocytes were stimulated with various concentrations of PPS, f-PPS or dextran for 72 h. 3H-thymidine was added to the cultures at the last 8 h of incubation and the results are expressed as 3H-thymidine incorporation (cpm).

Table 1 Quantitation of endotoxin activity in PPSa.

PPS

Concentration (lg/ml)

Endotoxin (Eu/ml)

100

0.054

a

LAL chromogenic assay was performed to determine LPS (endotoxin) activity in PPS (100 lg/ml). A standard curve was prepared for each experiment using the standard endotoxin. Table 2 Effect of PPS on production of IL-1 and TNF-a by C3H/HeN mouse peritoneal macrophages. 1

Group

IL-1b/ lg L

RPMI-1640 RPMI-1640 + polymyxin B Dextran Dextran + polymyxin B LPS LPS + polymyxin B PPS PPS + polymyxin B

0.03 ± 0.003 0.03 ± 0.003 0.05 ± 0.007 0.05 ± 0.005 0.49 ± 0.059 0.07 ± 0.047a 0.36 ± 0.068 0.31 ± 0.059b

TNF-a/ lg L

1

0.03 ± 0.005 0.03 ± 0.005 0.06 ± 0.009 0.05 ± 0.007 0.38 ± 0.041 0.08 ± 0.035a 0.27 ± 0.053 0.26 ± 0.052b

Results are expressed as means ± SEM and are representative of three independent experiments. a Significant difference at P < 0.01 between LPS and LPS + polymyxin B treatments. b Significant difference at P > 0.05 between PPS and PPS + polymyxin B treatments.

or without PPS for 48 h. BMDCs treated with LPS (100 ng/ml) or PPS (50 lg/ml) showed increased expression of CD86 (Fig. 2). The immunostimulatory activity of PPS was similar (albeit much less potent) to that of LPS, the strongest DC activator [13,14]. It was, therefore, of importance to exclude the possibility that the PPS preparations used in our study were contaminated with LPS. As shown in Table 1, the endotoxin level of 100 lg/ml of PPS preparation was 0.054 EU/ml (<0.1 EU/ml of endotoxin activity, corresponding to 8–25 pg/ml LPS depending on the LPS type), indicating that the level of LPS contamination in the PPS was negligible. 3.4. PPS increases IL-12 p40 and IL-10 production by BMDCs Central to the development of protective immune responses is the ability of a vaccine antigen to interact with and activate professional antigen presentation cells, i.e. DCs, the APCs most able to activate naïve T cells and initiate the development of acquired immune responses. Therefore, we determined the ability of PPS to stimulate DCs and induce a response. BMDCs were incubated with 12.5– 100 lg/ml PPS and the levels of IL-12 p40 and IL-10 in the culture supernatants were measured using cytokine specific ELISAs. As shown in Fig. 3, stimulation of BMDCs with PPS resulted in a dosedependent increase in both IL-12 p40 and IL-10. 3.5. PPS downregulates BMDC endocytic activity Immature BMDCs capture and process antigens via their high endocytic capacity. They lose this activity and mature into potent immunostimulatory APC during differentiation. The uptake of f-dextran is known to be maximal in immature BMDCs. Previous studies showed that the endocytic capacity of BMDCs is suppressed by LPS during the maturation process. Thus, we tested whether PPS affected the uptake of f-labeled dextran by BMDCs. We found that f-dextran uptake was reduced when BMDCs matured in response to PPS (Fig. 4). 3.6. Enhancement of T cell activation by PPS-treated BMDCs Mature BMDCs have the capacity to induce proliferation in allogeneic T cells to a greater extent than immature BMDCs

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10

5

28%

4

10

10

2

10

1

10

0

10 0 1 2 3 4 5 10 10 10 10 10 10

3

10

10 0 1 2 3 4 5 10 10 10 10 10 10

5

30%

4

10

3

10

2

10

1

10

0

10 0 1 2 3 4 5 10 10 10 10 10 10

PPS 25 10

2 1

3 2

33%

10

10 0 1 2 3 4 5 10 10 10 10 10 10

46%

4

10

3

10

2

10

1

10

0

10 0 1 2 3 4 5 10 10 10 10 10 10

3 2 1

10

0

10

4

10

1

PPS 100 5

5

10

10

10

0

PPS 50

10

10

56%

10

3

10

5

10

4

10

10

CD86

43%

4

10

PPS 12.5

dextran

medium 5

10 0 1 2 3 4 5 10 10 10 10 10 10

0

LPS 5

10

47%

4

10

3

10

2

10

1

10

0

10 0 1 2 3 4 5 10 10 10 10 10 10

CD11c Fig. 2. PPS enhance the expression of surface molecules of BMDCs. DC were untreated (medium), or were stimulated for 48 h with PPS (12.5, 25, 50 and 100 lg/ml), 50 lg/ml dextran or 100 ng/ml LPS on day 5, then the BMDCs were stained with PE-conjugated anti-CD86 mAb and FITC-conjugated anti-mouse CD11c mAb for 30 min. Stimulation of BMDC with PPS for 48 h resulted in a dose-dependent up-regulation of the CD86, and the 50 lg/ml was regarded as the optimal dose because the regulation effect of 100 lg/ml did not enhance significantly.

[15]. In BMDCs, we found that PPS up-regulated cell-surface markers and increased IL-12 production. To test whether this maturation is sufficient to promote the activation of naïve T cells, BMDCs were treated with LPS or PPS. These cells were then used to activate allogeneic, naïve T cells. As expected, BMDCs treated with PPS were able to stimulate allogeneic lymphocytes very efficiently (Fig. 5).

3.7. PPS induces IL-12 synthesis through TLR4 In order to verify whether TLR4 was involved in the interaction of DCs with PPS, neutralization experiments were performed. Immature DCs were preincubated with 20 lg/ml anti-TLR2, -TLR4 or -CR3 antibodies before treatment with PPS. The results showed that the addition of anti-mouse TLR4 to DC blocked PPS-induced IL-12 p40 production by around 53%, but the addition of antiTLR2 or -CR3 mAbs failed to inhibit PPS-induced IL-12 p40 produc-

tion (Fig. 6). These results suggested that TLR4 is involved in PPS activation of murine DCs.

3.8. Specific binding of PPS to DC In order to identify DC expressing specific receptor(s) for PPS and dextran were conjugated with fluoresceinamine using the CNBr-activation method. BMDCs were stained with f-PPS or f-dextran and then subjected to flow cytometric analysis. BMDCs were positively stained by f-PPS (Fig. 7). The mean fluorescence intensity (MFI) of BMDCs did not increase significantly when f-labeled PPS were used at higher concentrations (data not shown). Receptor-specific staining was confirmed using 1 lg/ml of each fluorescence-labeled polysaccharide by showing that staining was blocked in the presence of a 100-fold molar excess (100 lg/ml) of unlabeled PPS (Fig. 7A). Anti-TLR4 (Fig. 7A), but not anti-TLR2 and CR3 (Fig. 7B) mAbs also significantly blocked all BMDCs staining by

X. Li et al. / Cellular Immunology 265 (2010) 50–56

A

IL-12p40 concentration (ng/ml)

54

25 20 15

*

*

10

*

5 0 Medium dextran LPS

12.5

25

50

100

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Fig. 5. PPS enhances T cell response. Immature BMDCs were stimulated with LPS (100 ng/ml) or PPS (50 lg/ml) for 48 h. Allogeneic T cell proliferation was measured after 3 days of coculture with BMDCs. cpm, Counts per minute.

*

*

50

100

* Medium dextran LPS

12.5

25

25

IL-12 p40 concentration (ng/ml)

B

IL-10 concentration (ng/ml)

PPS (µg/ml)

PPS (µg/ml)

15 10

*

5

no Ab

-TLR4

-TLR2

Medium dextran LPS

IgG1

0

-CR3

Fig. 3. Effects of the PPS on the IL-12 and IL-10 production. After the development of immature DC (on day 5), 2  105 cells/ml were cultured with medium alone, dextran, LPS or different concentrations of PPS for 48 h at 37 °C. IL-12 p40 (A) and IL-10 (B) in culture supernatant was determined by ELISA. Statistical analysis concerns unstimulated (dextran) versus stimulated DC. * P < 0.05. Results are expressed as the mean ± SEM of triplicate from of three representative experiments.

20

PPS (50µg/ml) Fig. 6. Neutralization with TLR4 mAb inhibits the synthesis of IL-12 p40 in PPStreated BMDCs. Day 5 BMDCs were pre-treated with 20 lg/ml anti-TLR2, TLR4, CR3 and IgG1 antibodies separately for 1 h before adding PPS (50 lg/ml). After 24 h, the concentrations of IL-12 p40 concentration in the culture supernatants were determined by ELISA. Significant difference between BMDC treated with antibodies and no antibodies (no Ab) is indicated by P < 0.05(*). Results are expressed as the mean ± SEM of triplicate from of three representative experiments.

f-PPS. In contrast, dextran did not exhibit any significant inhibitory effects (data not shown). 4. Discussion

Recently, several studies demonstrated the immunomodulatory effects of PPS. However, little is known about the molecular mechanisms responsible for the regulation of DCs in their activation and maturation states by PPS. This study is the first to show PPS-induced phenotypic and functional changes in BMDCs. First, we identified the immunostimulatory activities of PPS. As shown in Fig 1, PPS significantly stimulates the proliferation of mouse splenocytes. The possibility of endotoxin contamination was ruled out using a LAL assay. We demonstrated that PPS up-regulated the expression of CD86 and

Relative cell number

DC maturation is a crucial step in the initiation of adaptive immune responses [16]. This process is regulated by various extracellular stimuli, including cytokines, bacterial products and membrane-bound ligands. BMDC maturation is accompanied by changes in their morphologic, phenotypic and functional properties [17,18]. Maturation of DCs is characterized by a decreased antigen-processing capacity, increased cell-surface expression of MHC class II molecules and the co-stimulatory molecules CD80, CD86 and CD40, and the secretion of IL-12, which primes the stimulation of T lymphocyte growth and differentiation [19,20].

medium

80

LPS

80

60

60

60

40

40

40

20

20

20

0

0 0

1

2

3

4

5

10 10 10 10 10 10

PPS

80

0 0

1

2

3

4

5

10 10 10 10 10 10

0

1

2

3

4

5

10 10 10 10 10 10

Fluorescence intensity Fig. 4. PPS on the endocytotic capacities of BMDCs. Immature BMDCs were cultured with medium, LPS, or PPS for 48 h, and cells were then incubated with f-dextran for 1 h at 4 °C (dotted lines) or 37 °C (solid lines).

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X. Li et al. / Cellular Immunology 265 (2010) 50–56

Relative cell number

A

B

100 PPS +f-PPS

75

100 -TLR2 +f-PPS

75

f-PPS

50

50 -TLR4 +f-PPS

25

-CR3 +f-PPS

25 0

0 0

10

1

10

2

10

3

10

4

10

5

10

0

10

1

10

2

10

3

10

4

10

5

10

Fluorescence intensity Fig. 7. TLR4-dependent staining of BMDCs with f-PPS. f-PPS staining of BMDCs was blocked by 100 lg/ml unlabeled PPS (A) and 200 lg/ml anti-TLR4 but not by anti-TLR2 and CR3 Ab (B). Green line histogram: PPS + f-PPS; black line histogram:f-PPS; blue line histogram:a-TLR4+f-PPS; red line histogram:a-TLR2+f-PPS; yellow line histogram:a-CR3+fPPS; grey-filled histogram: f-dextran negative control.

CD11c in a dose-dependent manner, suggesting that PPS induces phenotypically mature BMDCs. Recently, two DC subsets, DC1 and DC2, were identified. DC1 DC secretes IL-12 and induces the differentiation of T helper cell type Th0 T cells into Th1 cells; however DC2 DC induces Th0 T cells to differentiate into Th2 cells. IL-12 is a 70 kDa heterodimeric cytokine composed of disulfide-linked subunits of 35 and 40 kDa, respectively. IL-12 plays an important role as the link between the innate and adaptive immune systems. It is the most powerful stimulator of NK cell activation and can induces both IFN-c production and lytic activity [21,22]. In addition, IL-12 polarizes the immune system towards a primary Th1 response [23]. In this study, we found that PPS can significantly induce IL-12 production by BMDCs. The results show that PPS induces DC maturation and differentiation into DC1, and then activates NK and Th1 cells to enhance immune responses and anti-tumor effects. The major difference between DCs and other APCs is that DCs can activate naïve T cells. The MLR result demonstrated that PPS can activate CD4+CD45RA+ T cells (naïve T cells). PPS also reduces the phagocytic ability of BMDCs. These results show that PPS induces both phenotypic and functional maturation of BMDCs. The bioactivity of natural polysaccharides relies on polysaccharide-receptor interactions. Further research on polysaccharide receptors will be helpful in elucidating the mechanisms underlying immunomodulation by polysaccharides. It is reported that TLR2, TLR4, CR3 and dectin-1 are the receptors of natural polysaccharides [24,25]. TLR4 is present on the surface of many tissue cells and recognizes many kinds of pathogen associated molecular patterns (PAMPs), including LPS. Thereby, it initiates innate immune responses during the early stages of infection by pathogens. In addition, TLR4 can promote cytokine production, the expression of related immune molecules on the surface of immune cells, and immune cell maturation and activation. Thus, TLR4 plays an important role in regulating both anti-inflammatory and immune responses. Several natural polysaccharides, for example, PS-G (polysaccharide from Ganoderma lucidum) promote the activation and maturation of BMDCs and macrophages via the TLR4-modulated NF-jB pathway [26]. Therefore, in this study, the role of TLR4 in PPS-induced BMDC activation was investigated. We demonstrated that anti-TLR4 mAb significantly blocks PPS-induced IL-12 p40 production, but the addition of anti-TLR2 or -CR3 mAbs failed to inhibit PPS-induced IL-12 p40 production. These results show that TLR4 is involved in PPS-mediated DC maturation. Also, anti-TLR4 inhibits the binding of f-PPS to DCs, further demonstrating that TLR4 plays a critical role in the process PPS-triggered DC maturation.

However, it is likely that PPS recognition receptors unrelated to TLR4 receptors are also expressed on DC surfaces, because antiTLR4 mAb showed only partial inhibition of IL-12 production and did not completely inhibit f-PPS binding to pMus. Experiments aimed at investigating polysaccharide-membrane protein co-precipitation and proteomics are ongoing in our laboratory. In summary, our data show that PPS enhances the phenotypic and functional maturation of DCs via TLR4. These results will contribute to our understanding of Zhu Ling-mediated immunomodulatory activity. Acknowledgments This work was supported by grants from the Scientific Research Foundation of Wenzhou Medical College (QTJ07021) and Wenzhou Science and Technology Bureau Grant (Y20080126).

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