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Activation of dendritic cells by low molecular weight oyster polysaccharides
International Immunopharmacology 44 (2017) 183–190
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Activation of dendritic cells by low molecular weight oyster polysaccharides Ming Zhong a,⁎, Cheng Zhong b, Tingting Wang c, Pei Hu d, Guanghui Wang a, Rujing Ren a, Jing Zhang a, Huijei Gao a, Wen Cui a, Wenjuan Duan e, Jiantu Che f a
Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao 276826, China Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Yamadaoka2-2, Suita, Osaka 565-0871, Japan People's Hospital of Rizhao, Taian Road 126, Rizhao 276826, China d Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai, China e Shandong Analysis and Test Center, Keyuan Road 19, Jinan 250014, China f S&V Biological Science and Technology Co., Ltd, Heying Road 8, Nanshao, Changping, Beijing 102200, China b c
a r t i c l e
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Article history: Received 25 July 2016 Received in revised form 27 December 2016 Accepted 10 January 2017 Available online 19 January 2017 Keywords: Polysaccharides Oyster Dendritic cells Maturation MAPK
a b s t r a c t Dendritic cells play a primary role in antigen presentation to CD4+ T cells, which initiate acquired immune responses. Therefore, determining positive modulators of dendritic cell activation to improve therapeutic approaches for cancer treatment might be useful. We here investigated the effects of low molecular weight oyster polysaccharides (LMW-OPS) on bone marrow-derived dendritic cells (BMDCs) obtained from mice. LMW-OPS increased the surface expression of major histocompatibility complex class II (MHC-II), CD40 and CD86 in BMDCs and induced the secretion of tumour necrosis factor (TNF)-α and interleukin (IL)-12, which were significantly decreased in the BMDCs derived from MyD88−/− mice but not from the lipopolysaccharideresistant C3H/HeJ mice. BMDCs treated with LMW-OPS augmented allogeneic CD4+ T cell expansion and enhanced secretion of IL-2 and interferon (IFN)-γ but not IL-4. LMW-OPS induced significant increases in ERK and p38 MAPK phosphorylation, but not c-Jun N-terminal kinase (JNK) phosphorylation, in BMDCs. Our results indicate that, in part, LMW-OPS can induce maturation of BMDCs in a MyD88-dependent and Toll-like receptor (TLR) 4-independent manner. LMW-OPS may enhance acquired immunity by modulating the function of dendritic cells. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Dendritic cells (DCs), which function as professional antigen presenting cells to CD4+ T cells, are recognized as an attractive target in cancer immunotherapy because of their role in inducing and modulating effective immune responses against tumour cells [1]. DCs undergo a process of differentiation known as maturation. This process significantly improves their capacity for antigen processing and presentation [2–4]. The general features of DC maturation are well understood and involve the Abbreviations: LMW-OPS, low molecular weight oyster polysaccharides; DCs, dendritic cells; IFN, interferon; BMDCs, bone marrow-derived dendritic cells; IL, interleukin; MHC, major histocompatibility complex; TLR, Toll-like receptor; TNF, tumour necrosis factor; ERK, extracellular signal regulated kinase; MAPK, mitogenactivated protein kinase; JNK, c-Jun N-terminal kinase; GM-CSF, granulocyte macrophage colony stimulating factor; MyD88, myeloid differentiation factor 88; i DCs, immature DCs; LMW-OPS-DCs, LMW-OPS-treated DCs; LPS-DCs, LPS-treated DCs. ⁎ Corresponding author. E-mail address: [email protected] (M. Zhong).
http://dx.doi.org/10.1016/j.intimp.2017.01.018 1567-5769/© 2017 Elsevier B.V. All rights reserved.
redistribution of MHC-II molecules from the intracellular compartments to the cell surface, increased co-stimulatory molecules expression, such as CD86 and CD80, and the release of cytokines, such as TNF-α and IL12 [5–8]. However, DCs distributed in the tumour microenvironment are generally regarded as immature DCs, which are unable to fully induce anti-tumour responses but can induce T cell tolerance or anergy [9]. Numerous attempts have been made to discover organic substances that could contribute to the induction of DC maturation. Plant and medicinal fungus-derived polysaccharides that enhance anti-tumour effects by boosting host immune functions, such as the polysaccharides derived from Phellinus linteus and Cordyceps militaris, have been shown to induce phenotypic maturation and IL-12 secretion in DCs [10,11]. In contrast to polysaccharides derived from plants and medicinal fungi, little evidence is available concerning DC maturation induced by polysaccharides derived from animal origins. As a dietary supplement, oyster extracts are widely consumed in many countries. Recent studies on the pharmacological properties of oyster extracts and their constituents such as oyster polysaccharides have shown various
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biological effects including anti-tumour and immunostimulatory activities [12,13]. In this study, we focused on the effects of low molecular weight oyster polysaccharides (LMW-OPS) on DC maturation. 2. Materials and methods 2.1. Reagents Recombinant mouse GM-CSF was provided by PeproTech Inc. (Rocky Hill, NJ). Fluorescently labelled anti-mouse monoclonal antibodies
(CD11c-PE, MHC-II-FITC, CD86-FITC, CD80-FITC and CD40-FITC) and cytokine ELISA assay kits (IL-12, TNF-α, IL-2, IFN-γ and IL-4) were obtained from eBioscience (San Diego, CA). Anti-phosphoERK1/ERK2, anti-phospho-p46 c-Jun N-terminal kinase (JNK)/p54 JNK and anti-phospho-p38 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-p46 JNK, anti-p38 MAPK and anti-ERK1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Lipopolysaccharide (LPS) from E. coli 055:B5, and [3H]thymidine were obtained from Sigma Chemical Co. (St. Louis, MO).
Fig. 1. Maturation of DCs in vitro and in vivo induced by LMW-OPS. For in vitro DC maturation assays, BMDCs were generated from C57BL/6 mice and treated with LMW-OPS (0.1–1 mg/ml) for 24 h. The surface expression of MHC-II, CD86, and CD40 in CD11c+ cells was analysed using flow cytometry (A). The mean fluorescence intensity (MFI) of each marker is presented (B– D). For determination of in vivo effect of LMW-OPS, spleen cells were isolated from the mice intraperitoneally administered with LMW-OPS (100 mg/kg and 200 mg/kg, 6 consecutive days) and the surface expression of MHC-II and CD86 in the CD11c+ splenic DCs was analysed using flow cytometry (E). The mean fluorescence intensity (MFI) of each marker is presented (F, G). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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Fig. 2. Effect of polymyxin B (PMB) on LMW-OPS- or LPS-induced DC maturation. BMDCs were generated from C57BL/6 mice and treated with or without 10 μg/ml of PMB for 2 h, followed by treatment with LMW-OPS (1 mg/ml) or LPS (1 μg/ml) for 24 h. The surface expression of MHC-II and CD86 in CD11c+ cells was analysed using flow cytometry (A, B). The mean fluorescence intensity (MFI) of each marker is presented (C, D). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
Fig. 3. Effect of LMW-OPS on C3H/HeJ DC maturation. BMDCs were generated from C3H/HeJ mice and treated with LMW-OPS (0.3 and 1 mg/ml) for 24 h. The surface expression of MHC-II and CD86 in CD11c+ cells was analysed by flow cytometry (A). The mean fluorescence intensity (MFI) of each marker is presented (B, C). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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2.2. Preparation of LMW-OPS Fresh oysters, Crassostrea gigas, were obtained from a hanging culture bed in the Huanghai Sea of Rizhao, China. Oysters were extracted with 10 volumes of water for 1.5 h at 120 °C, and the mixture was filtered by a 20 mesh nylon sieve. The harvested filtrate was sterilized and spray-dried. Protein removal was performed using the Sevag method [14], and the resultant materials was dissolved by water and then added with 95% EtOH to produce a final concentration of 30% (v/v), after centrifuged, the supernatant was collected and concentrated. The concentrate was added with 95% EtOH to obtain a final concentration of 50% (v/v) and then centrifuged to gain the precipitate. The precipitate are dried in vacuum at 45 °C and defined as oyster polysaccharides (OPS). The OPS were further fractionated by freezing and thawing to remove the insoluble materials, the soluble fraction was purified by using
Sepharose CL-6B gel filtration chromatography. The main purified products are referred to as LMW-OPS. Dynamic light scattering (DLS) and high performance gel permeation chromatography (HPGPC) indicated that the LMW-OPS (MW = 1980–2630 Da) were a homogeneous polysaccharide (Fig. S1). Gas chromatography–Mass Spectrometry (GC–MS) and FT-IR analysis showed that the LMW-OPS were composed of α-Dglucopyranose (Fig. S2).
2.3. Mice C57BL/6, C57BL/6 MyD88−/−, C3H/HeN, C3H/HeJ and BALB/c mice (6–12 weeks of age) were obtained from Shandong University Laboratory Animal Centre (Jinan, China). All mice were guaranteed free of particular pathogens in the Laboratory Animal Centre at Jining Medical
Fig. 4. IL-12 and TNF-α production in DCs. BMDCs were generated from wild type, MyD88−/−, C3H/HeN, and C3H/HeJ mice and then treated with LMW-OPS or LPS (1 μg/ml) for 24 h. The levels of IL-12 (A, B, C) and TNF-α (D, E, F) in the culture supernatants were measured using an ELISA. The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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College. Approval of the animal care protocols were obtained from the Animal Care and Use Committee at Jining Medical College. 2.4. Generation of bone marrow-derived dendritic cells (BMDCs) Bone marrow cells were flushed from the tibias and femurs of C57BL/6, C57BL/6 MyD88−/−, C3H/HeJ and C3H/HeN mice. After treatment with a mixture of A solution (0.83% of ammonium chloride) 9: B solution (0.17 M Tris-HCl buffer) 1, pH 7.2, for red blood cells depletion, bone marrow cells (2 × 106 cells/ml) were seeded into 12 well culture plates in RPMI 1640 medium containing 10% heat-inactivated foetal calf serum (FCS), 100 μg/ml of streptomycin, 100 U/ml of penicillin G and 2 mM L-glutamine (complete RPMI 1640 medium) in the presence of 15 ng/ml of GM–CSF. The medium was changed every 2 days. On day 6, loosely adherent and non-adherent cells were collected by vigorous pipetting and used as BMDCs. Flow cytometry showed that N95% of the BMDCs were CD11c+ and CD3−/B220−. 2.5. Phenotypic characterization of DCs For in vitro phenotypic characterization of DCs, BMDCs were seeded at a concentration of 2 × 106 cells in 6 well culture plates (Corning, Cultek, Madrid, Spain) and incubated in 4 ml of complete RPMI 1640 medium containing 15 ng/ml of GM-CSF with or without LMW-OPS at 37 °C for 24 h in a fully humidified atmosphere containing 5% CO2. The cells were resuspended in PBS containing 2% FBS and stained at 4 °C for 30 min with FITC-labelled anti-MHC-II, anti-CD86, anti-CD40, or anti-CD80 plus PE-labelled CD11c antibodies. The surface molecule expression level of BMDCs was analysed using flow cytometry (BD FacsCanto II, USA). The culture supernatants were obtained and stored
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at − 80 °C until cytokine determination (TNF-α and IL-12) using the ELISA kits. For in vivo determination of the effect of LMW-OPS on DC maturation, C57BL/6 mice were intraperitoneally administered 100 mg/kg and 200 mg/kg of LMW-OPS in 0.2 ml/head of saline or saline alone (control) for 6 consecutive days. Erythrocyte-depleted spleen cells were isolated and stained with FITC-labelled anti-MHC-II or anti-CD86 plus PE-labelled CD11c antibodies. The surface molecule expression level of splenic DCs was analysed using flow cytometry.
2.6. Mixed lymphocyte reaction T cells were enriched from the spleen of BALB/c mice by negative selection using a mouse T Lymphocyte Enrichment Set (BD Biosciences Pharmingen). Briefly, murine spleen cells were treated with biotin-conjugated specific antibodies against CD11c, B220, and GR-1 and mixed with BD IMag streptavidin particles. Negative selection was then performed on a BD IMagnet according to the manufacturer's protocol to enrich the unlabelled T cells. The purity was typically N 95%. To label the cells with CFSE (Invitrogen), the enriched T cells were suspended in phenol red and FCS-free medium, and CFSE was added at a final concentration of 0.5 μM. The cells were incubated at 37 °C for 8 min and washed two times with PBS containing 5% FCS and one time with complete RPMI 1640 medium, and then the cells were resuspended in complete RPMI 1640 medium. The BMDCs were generated from C57BL/6 mice and treated with or without LMW-OPS (1 mg/ml) for 24 h at 37 °C, and then the cells were washed two times with complete RPMI 1640 medium and co-cultured with CFSE-stained T cells in U-bottom 96 well plates (DCs:T cells = 1:10). The cells were cultured for 72 h at 37 °C in 5% CO2 and then stained with PE-labelled anti-mouse CD4
Fig. 5. Proliferation of allogeneic CD4+ T cells induced by LMW-OPS-treated DCs. BMDCs were generated from C57BL/6 mice and treated with LMW-OPS (1 mg/ml) or LPS (1 μg/ml) for 24 h. The proliferation of CD4+ T cells was evaluated with co-cultured CFSE-labelled BALB/c mouse-derived T cells (DCs:T = 1:10) and using flow cytometry three days after the co-culture (A, B). The proliferation of T cells was determined by measuring [3H]thymidine uptake in three days after co-cultured BMDCs with allogeneic T cells (C). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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antibody. CD4+ T cell expansion was detected by CFSE dilution using a flow cytometer. The cell culture supernatants were obtained and stored at −80 °C until IFN-γ and IL-2 determination using the ELISA kits. For T cell proliferation assays, the BMDCs from C57BL/6 mice were treated with 20 μg/ml mitomycin C for 2 h. The mitomycin C-treated BMDCs (4 × 104 cells) were added to 4 × 105 T cells in U-bottom 96 well plates (DCs:T cells = 1:10). The cells were co-cultured for 72 h at 37 °C in 5% CO2, and the allogenic T cells from BALB/c mice were pulse-labelled with [3H]thymidine (1.0 μCi/well, 5.0 Ci/mmol) for 16 h and then harvested on glass fiber filters using an automated cell harvester. The amount of [3H]thymidine incorporated was measured using a liquid scintillation counter. 2.7. Western blotting For Western blot analysis, control or LMW-OPS-treated DCs were lysed, and proteins from cell lysates were loaded onto 10% SDS-PAGE gels. Western blotting analysis was conducted by using the following primary antibodies: anti-phospho-p46 JNK/p54 JNK, anti-p46 JNK, anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-ERK1/ERK2 and anti-ERK1. The immunoblots were visualized using ECL detection reagents (Amersham). 2.8. Data analysis The results are expressed as the means and SDs of more than three independent experiments. Student's t-test was used to analyse data in
two groups. Tukey's test (SPSS) was following multiple comparisons analysed by one-way ANOVA. A p value b 5% was regarded as statistically significant.
3. Results 3.1. LMW-OPS-induced phenotypic maturation of DCs LMW-OPS increased the surface expression of MHC-II (Fig. 1A and B), CD86 (Fig. 1A and C), and CD40 (Fig. 1A and D), which are markers of DC maturation, in a dose-dependent manner in BMDCs. LMW-OPS also significantly enhanced the expression of MHC-II and CD86 in splenic DCs after intraperitoneal administration in vivo (Fig. 1E, F and G). In the in vivo experiment, no difference was observed between control mice and mice treated with LMWOPS in final body weights gains (data not shown). To exclude the possibility that LPS contamination of the LMW-OPS produced the up-regulation of these markers, we performed two experiments; one experiment used polymyxin B (PMB), which can bind to and neutralize LPS, and the other experiment used BMDCs obtained from C3H/HeJ mice, which express mutated Toll-like receptor 4 (TLR4). The LMW-OPS-induced up-regulation of MHC-II and CD86 expression was not influenced by PMB, which blocked the effects of LPS (Fig. 2A–D). LMW-OPS significantly increased MHC-II and CD86 expression on the surface of BMDCs obtained from C3H/HeJ mice (Fig. 3A–C).
Fig. 6. LMW-OPS-mediated enhancement of Th1 cytokine production induced by allogeneic responses. BMDCs were generated from C57BL/6 mice and treated with LMW-OPS (1 mg/ml) or LPS (1 μg/ml) for 24 h and then co-cultured with BALB/c mouse-derived T cells (DCs:T = 1:10). The levels of IL-2 (A), IFN-γ (B), and IL-4 (C) released during the 3 h of co-culture were measured. The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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3.2. Induction of IL-12 and TNF-α in BMDCs stimulated with LMW-OPS LMW-OPS also induced cytokine production in BMDCs. IL-12 and TNF-α, which were significantly decreased in the BMDCs obtained from MyD88−/− mice, were induced by LMW-OPS in a dose-dependent manner (Fig. 4A, B, D and E). LPS did not induce the secretion of IL-12 and TNF-α in the BMDCs obtained from TLR4 mutant C3H/HeJ mice (data not shown). LMW-OPS-induced release of these cytokines was not changed in the BMDCs of C3H/HeJ mice (Fig. 4C and F), and not impacted by polymyxin B in the BMDCs of C3H/HeN mice (data not shown), indicating that TLR4 is not involved in the action of LMW-OPS. 3.3. LMW-OPS-mediated enhancement of T cell expansion and Th1 cytokine release in mixed lymphocyte reactions We investigated whether LMW-OPS-activated BMDCs could enhance T cell responses. Proliferation of T cells (Fig. 5C) and CD4+ T cells (Fig. 5A and B) induced by mixed lymphocyte reactions was enhanced in BMDCs pre-treated with LMW-OPS or LPS (Fig. 5). Allogeneic secretion of IL-2 (Fig. 6A) and IFN-γ (Fig. 6B) was also significantly increased in BMDCs pre-treated with LMW-OPS. In contrast, production of IL-4 was not affected (Fig. 6C). 3.4. Increased phosphorylation of MAPKs in BMDCs treated with LMW-OPS To understand the underlying mechanisms of the changes in BMDCs, we investigated changes in the phosphorylation levels of MAPKs, including ERK, JNK, and p38 MAPK, in BMDCs stimulated with LMWOPS. We observed that both ERK1/2 and p38 MAPK phosphorylation
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levels were significantly up-regulated at 5 and 15 min after LMW-OPS treatment (Fig. 7A–C). However, LMW-OPS had no effect on JNK phosphorylation (Fig. 7A and D). 4. Discussion Studies have shown that the level of DC maturation determines the type of DC-induced immune response. The expression levels of MHCII, CD86 and CD40 are much higher in mature DCs, which have a strong ability to initiate T cell activation. However, immune tolerance can be induced if the MHC-II and CD86 expression on the surface of DCs is decreased in the tumour microenvironment [15,16]. Previous studies have found that immunomodulators, such as plant and medicinal fungus-derived polysaccharides, induce DC maturation during tumour immunotherapy [10,11,17,18]. In our study, we found that low levels of expression of MHC-II, CD86 and CD40 in immature BMDCs were significantly up-regulated by LMW-OPS (Fig. 1–3), we also observed that MHC-II and CD86 expression in splenic DCs were enhanced by in vivo treated with LMW-OPS. Our discovery indicates that LMW-OPS can induce DC maturation. T cell-mediated immune responses, which play an important role in anti-tumour immunity, and particularly their secretion of various cytokines are largely regulated by DCs [19,20]. LMW-OPS stimulated the secretion of TNF-α and IL-12, which are involved in Th1 skewing. Furthermore, LMW-OPS treatment drastically augmented the secretion of IL-2 and IFN-γ in BMDCs during allogeneic responses. These results strongly indicate that LMW-OPS induce T-cell polarization to Th1. Current research shows that the TLR signalling pathway has a crucial function in DCs by initiating the immune response, and ligation of TLRs
Fig. 7. Activation of MAPKs by LMW-OPS. BMDCs were generated from C57BL/6 mice and treated with or without LMW-OPS (1 mg/ml) for the indicated periods. Phosphorylation of ERK (B), p38 MAPK (C), and JNK (D) was measured by immunoblot analysis. The image (A) is representative of five independent experiments. The values are presented as the means ± SD (n = 5). A value of *p b 0.05 is regarded as statistically significant.
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can lead to rapid DC activation and generate Th1-polarizing DCs [21– 26]. Studies have also shown that except for TLR3, MyD88 is a signal transducing adaptor protein that activates the TLR signalling pathway [27,28]. Therefore, we tested whether the mechanism of LMW-OPS-induced DC maturation was related to the MyD88 signalling pathway. As shown in Fig. 4B and D, LMW-OPS-induced release of IL-12 and TNF-α was significantly reduced in BMDCs obtained from the MyD88−/− mice, but not changed in the C3H/HeJ mice (Fig. 4D and F), which indicates that LMW-OPS-induced DC maturation or activation relies on TLRs/ MyD88 signalling in a TLR4-independent manner. However, the secretion of TNF-α and IL-12 was not completely eliminated in BMDCs obtained from MyD88−/− mice, suggesting that LMW-OPS can also interact with other signal transduction pathways such as the TRIF pathway [29,30]. The phosphorylation levels of ERK and P38 MAPK were up-regulated by LMW-OPS. Studies have shown that TLR3 uses TRIF adaptor molecules [29–33], such as the TLR3/TRIF/MAPK signal transduction pathway [33]. However, the secretion of IL-12 and TNF-α in immature BMDCs by polysaccharides such as β-glucan extracts is dependent on the dectin-1 pathway [34]. Therefore, we are currently further analysing the mechanism of LMW-OPS-induced DC maturation in the TLR-dependent and independent pathways. Recently, we found that oral administration of the water-ethanol extract of oysters could suppress tumour progression in tumour-bearing mice and activate natural killer cells in vitro (unpublished results). Furthermore, the enhanced effects of crude polysaccharides derived from oysters on antigen-specific Th1 immunity was also confirmed in vivo by Cheng et al. [13]. The present paper describes the purification of LMW-OPS, which are homogeneous polysaccharides, and the investigation of their effects on DC activation. The LMW-OPS-induced functional and phenotypic maturation of DCs is characterized by a robust increase in the expression of CD86, CD40 and MHC-II and the secretion of TNF-α and IL-12. This mechanism triggers the secretion of IL-2 and IFN-γ by naïve CD4+ T cells, which is a response of Th1 cells. Oysters are cultivated in many countries, and oyster dietary supplements are widely consumed. Our results suggest that LMW-OPS are active ingredients from oysters that affect dendritic cell-mediated immunity and can be utilized as a potential adjuvant to enhance the anti-tumour effects of DC based therapy. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2017.01.018. Acknowledgements This work was supported by the Shandong Provincial Natural Science Foundation, China (Grant No. ZR2014HM038). We thank Professor Satoshi Tanaka (Okayama University) for the valuable comments on this study. References [1] E. Gilboa, DC-based cancer vaccines, J. Clin. Invest. 117 (5) (2007) 1195–1203. [2] J. Banchereau, A.K. Palucka, Dendritic cells as therapeutic vaccines against cancer, Nat. Rev. Immunol. 5 (4) (2005) 296–306. [3] K. Palucka, J. Banchereau, Cancer immunotherapy via dendritic cells, Nat. Rev. Cancer 12 (4) (2012) 265–277. [4] K. Palucka, H. Ueno, J. Banchereau, Recent developments in cancer vaccines, J. Immunol. 186 (3) (2011) 1325–1331. [5] F.S. Lichtenegger, K. Mueller, B. Otte, et al., CD86 and IL-12p70 are key players for T helper 1 polarization and natural killer cell activation by Toll-like receptor-induced dendritic cells, PLoS One 7 (9) (2012), e44266. . [6] M.P. Longhi, C. Trumpfheller, J. Idoyaga, et al., Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant, J. Exp. Med. 206 (7) (2009) 1589–1602.
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Activation of dendritic cells by low molecular weight oyster polysaccharides Ming Zhong a,⁎, Cheng Zhong b, Tingting Wang c, Pei Hu d, Guanghui Wang a, Rujing Ren a, Jing Zhang a, Huijei Gao a, Wen Cui a, Wenjuan Duan e, Jiantu Che f a
Institute of Tumor Pharmacology, Jining Medical College, Xueyuan Road 669, Rizhao 276826, China Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, Yamadaoka2-2, Suita, Osaka 565-0871, Japan People's Hospital of Rizhao, Taian Road 126, Rizhao 276826, China d Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai, China e Shandong Analysis and Test Center, Keyuan Road 19, Jinan 250014, China f S&V Biological Science and Technology Co., Ltd, Heying Road 8, Nanshao, Changping, Beijing 102200, China b c
a r t i c l e
i n f o
Article history: Received 25 July 2016 Received in revised form 27 December 2016 Accepted 10 January 2017 Available online 19 January 2017 Keywords: Polysaccharides Oyster Dendritic cells Maturation MAPK
a b s t r a c t Dendritic cells play a primary role in antigen presentation to CD4+ T cells, which initiate acquired immune responses. Therefore, determining positive modulators of dendritic cell activation to improve therapeutic approaches for cancer treatment might be useful. We here investigated the effects of low molecular weight oyster polysaccharides (LMW-OPS) on bone marrow-derived dendritic cells (BMDCs) obtained from mice. LMW-OPS increased the surface expression of major histocompatibility complex class II (MHC-II), CD40 and CD86 in BMDCs and induced the secretion of tumour necrosis factor (TNF)-α and interleukin (IL)-12, which were significantly decreased in the BMDCs derived from MyD88−/− mice but not from the lipopolysaccharideresistant C3H/HeJ mice. BMDCs treated with LMW-OPS augmented allogeneic CD4+ T cell expansion and enhanced secretion of IL-2 and interferon (IFN)-γ but not IL-4. LMW-OPS induced significant increases in ERK and p38 MAPK phosphorylation, but not c-Jun N-terminal kinase (JNK) phosphorylation, in BMDCs. Our results indicate that, in part, LMW-OPS can induce maturation of BMDCs in a MyD88-dependent and Toll-like receptor (TLR) 4-independent manner. LMW-OPS may enhance acquired immunity by modulating the function of dendritic cells. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Dendritic cells (DCs), which function as professional antigen presenting cells to CD4+ T cells, are recognized as an attractive target in cancer immunotherapy because of their role in inducing and modulating effective immune responses against tumour cells [1]. DCs undergo a process of differentiation known as maturation. This process significantly improves their capacity for antigen processing and presentation [2–4]. The general features of DC maturation are well understood and involve the Abbreviations: LMW-OPS, low molecular weight oyster polysaccharides; DCs, dendritic cells; IFN, interferon; BMDCs, bone marrow-derived dendritic cells; IL, interleukin; MHC, major histocompatibility complex; TLR, Toll-like receptor; TNF, tumour necrosis factor; ERK, extracellular signal regulated kinase; MAPK, mitogenactivated protein kinase; JNK, c-Jun N-terminal kinase; GM-CSF, granulocyte macrophage colony stimulating factor; MyD88, myeloid differentiation factor 88; i DCs, immature DCs; LMW-OPS-DCs, LMW-OPS-treated DCs; LPS-DCs, LPS-treated DCs. ⁎ Corresponding author. E-mail address: [email protected] (M. Zhong).
http://dx.doi.org/10.1016/j.intimp.2017.01.018 1567-5769/© 2017 Elsevier B.V. All rights reserved.
redistribution of MHC-II molecules from the intracellular compartments to the cell surface, increased co-stimulatory molecules expression, such as CD86 and CD80, and the release of cytokines, such as TNF-α and IL12 [5–8]. However, DCs distributed in the tumour microenvironment are generally regarded as immature DCs, which are unable to fully induce anti-tumour responses but can induce T cell tolerance or anergy [9]. Numerous attempts have been made to discover organic substances that could contribute to the induction of DC maturation. Plant and medicinal fungus-derived polysaccharides that enhance anti-tumour effects by boosting host immune functions, such as the polysaccharides derived from Phellinus linteus and Cordyceps militaris, have been shown to induce phenotypic maturation and IL-12 secretion in DCs [10,11]. In contrast to polysaccharides derived from plants and medicinal fungi, little evidence is available concerning DC maturation induced by polysaccharides derived from animal origins. As a dietary supplement, oyster extracts are widely consumed in many countries. Recent studies on the pharmacological properties of oyster extracts and their constituents such as oyster polysaccharides have shown various
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biological effects including anti-tumour and immunostimulatory activities [12,13]. In this study, we focused on the effects of low molecular weight oyster polysaccharides (LMW-OPS) on DC maturation. 2. Materials and methods 2.1. Reagents Recombinant mouse GM-CSF was provided by PeproTech Inc. (Rocky Hill, NJ). Fluorescently labelled anti-mouse monoclonal antibodies
(CD11c-PE, MHC-II-FITC, CD86-FITC, CD80-FITC and CD40-FITC) and cytokine ELISA assay kits (IL-12, TNF-α, IL-2, IFN-γ and IL-4) were obtained from eBioscience (San Diego, CA). Anti-phosphoERK1/ERK2, anti-phospho-p46 c-Jun N-terminal kinase (JNK)/p54 JNK and anti-phospho-p38 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-p46 JNK, anti-p38 MAPK and anti-ERK1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Lipopolysaccharide (LPS) from E. coli 055:B5, and [3H]thymidine were obtained from Sigma Chemical Co. (St. Louis, MO).
Fig. 1. Maturation of DCs in vitro and in vivo induced by LMW-OPS. For in vitro DC maturation assays, BMDCs were generated from C57BL/6 mice and treated with LMW-OPS (0.1–1 mg/ml) for 24 h. The surface expression of MHC-II, CD86, and CD40 in CD11c+ cells was analysed using flow cytometry (A). The mean fluorescence intensity (MFI) of each marker is presented (B– D). For determination of in vivo effect of LMW-OPS, spleen cells were isolated from the mice intraperitoneally administered with LMW-OPS (100 mg/kg and 200 mg/kg, 6 consecutive days) and the surface expression of MHC-II and CD86 in the CD11c+ splenic DCs was analysed using flow cytometry (E). The mean fluorescence intensity (MFI) of each marker is presented (F, G). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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Fig. 2. Effect of polymyxin B (PMB) on LMW-OPS- or LPS-induced DC maturation. BMDCs were generated from C57BL/6 mice and treated with or without 10 μg/ml of PMB for 2 h, followed by treatment with LMW-OPS (1 mg/ml) or LPS (1 μg/ml) for 24 h. The surface expression of MHC-II and CD86 in CD11c+ cells was analysed using flow cytometry (A, B). The mean fluorescence intensity (MFI) of each marker is presented (C, D). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
Fig. 3. Effect of LMW-OPS on C3H/HeJ DC maturation. BMDCs were generated from C3H/HeJ mice and treated with LMW-OPS (0.3 and 1 mg/ml) for 24 h. The surface expression of MHC-II and CD86 in CD11c+ cells was analysed by flow cytometry (A). The mean fluorescence intensity (MFI) of each marker is presented (B, C). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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2.2. Preparation of LMW-OPS Fresh oysters, Crassostrea gigas, were obtained from a hanging culture bed in the Huanghai Sea of Rizhao, China. Oysters were extracted with 10 volumes of water for 1.5 h at 120 °C, and the mixture was filtered by a 20 mesh nylon sieve. The harvested filtrate was sterilized and spray-dried. Protein removal was performed using the Sevag method [14], and the resultant materials was dissolved by water and then added with 95% EtOH to produce a final concentration of 30% (v/v), after centrifuged, the supernatant was collected and concentrated. The concentrate was added with 95% EtOH to obtain a final concentration of 50% (v/v) and then centrifuged to gain the precipitate. The precipitate are dried in vacuum at 45 °C and defined as oyster polysaccharides (OPS). The OPS were further fractionated by freezing and thawing to remove the insoluble materials, the soluble fraction was purified by using
Sepharose CL-6B gel filtration chromatography. The main purified products are referred to as LMW-OPS. Dynamic light scattering (DLS) and high performance gel permeation chromatography (HPGPC) indicated that the LMW-OPS (MW = 1980–2630 Da) were a homogeneous polysaccharide (Fig. S1). Gas chromatography–Mass Spectrometry (GC–MS) and FT-IR analysis showed that the LMW-OPS were composed of α-Dglucopyranose (Fig. S2).
2.3. Mice C57BL/6, C57BL/6 MyD88−/−, C3H/HeN, C3H/HeJ and BALB/c mice (6–12 weeks of age) were obtained from Shandong University Laboratory Animal Centre (Jinan, China). All mice were guaranteed free of particular pathogens in the Laboratory Animal Centre at Jining Medical
Fig. 4. IL-12 and TNF-α production in DCs. BMDCs were generated from wild type, MyD88−/−, C3H/HeN, and C3H/HeJ mice and then treated with LMW-OPS or LPS (1 μg/ml) for 24 h. The levels of IL-12 (A, B, C) and TNF-α (D, E, F) in the culture supernatants were measured using an ELISA. The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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College. Approval of the animal care protocols were obtained from the Animal Care and Use Committee at Jining Medical College. 2.4. Generation of bone marrow-derived dendritic cells (BMDCs) Bone marrow cells were flushed from the tibias and femurs of C57BL/6, C57BL/6 MyD88−/−, C3H/HeJ and C3H/HeN mice. After treatment with a mixture of A solution (0.83% of ammonium chloride) 9: B solution (0.17 M Tris-HCl buffer) 1, pH 7.2, for red blood cells depletion, bone marrow cells (2 × 106 cells/ml) were seeded into 12 well culture plates in RPMI 1640 medium containing 10% heat-inactivated foetal calf serum (FCS), 100 μg/ml of streptomycin, 100 U/ml of penicillin G and 2 mM L-glutamine (complete RPMI 1640 medium) in the presence of 15 ng/ml of GM–CSF. The medium was changed every 2 days. On day 6, loosely adherent and non-adherent cells were collected by vigorous pipetting and used as BMDCs. Flow cytometry showed that N95% of the BMDCs were CD11c+ and CD3−/B220−. 2.5. Phenotypic characterization of DCs For in vitro phenotypic characterization of DCs, BMDCs were seeded at a concentration of 2 × 106 cells in 6 well culture plates (Corning, Cultek, Madrid, Spain) and incubated in 4 ml of complete RPMI 1640 medium containing 15 ng/ml of GM-CSF with or without LMW-OPS at 37 °C for 24 h in a fully humidified atmosphere containing 5% CO2. The cells were resuspended in PBS containing 2% FBS and stained at 4 °C for 30 min with FITC-labelled anti-MHC-II, anti-CD86, anti-CD40, or anti-CD80 plus PE-labelled CD11c antibodies. The surface molecule expression level of BMDCs was analysed using flow cytometry (BD FacsCanto II, USA). The culture supernatants were obtained and stored
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at − 80 °C until cytokine determination (TNF-α and IL-12) using the ELISA kits. For in vivo determination of the effect of LMW-OPS on DC maturation, C57BL/6 mice were intraperitoneally administered 100 mg/kg and 200 mg/kg of LMW-OPS in 0.2 ml/head of saline or saline alone (control) for 6 consecutive days. Erythrocyte-depleted spleen cells were isolated and stained with FITC-labelled anti-MHC-II or anti-CD86 plus PE-labelled CD11c antibodies. The surface molecule expression level of splenic DCs was analysed using flow cytometry.
2.6. Mixed lymphocyte reaction T cells were enriched from the spleen of BALB/c mice by negative selection using a mouse T Lymphocyte Enrichment Set (BD Biosciences Pharmingen). Briefly, murine spleen cells were treated with biotin-conjugated specific antibodies against CD11c, B220, and GR-1 and mixed with BD IMag streptavidin particles. Negative selection was then performed on a BD IMagnet according to the manufacturer's protocol to enrich the unlabelled T cells. The purity was typically N 95%. To label the cells with CFSE (Invitrogen), the enriched T cells were suspended in phenol red and FCS-free medium, and CFSE was added at a final concentration of 0.5 μM. The cells were incubated at 37 °C for 8 min and washed two times with PBS containing 5% FCS and one time with complete RPMI 1640 medium, and then the cells were resuspended in complete RPMI 1640 medium. The BMDCs were generated from C57BL/6 mice and treated with or without LMW-OPS (1 mg/ml) for 24 h at 37 °C, and then the cells were washed two times with complete RPMI 1640 medium and co-cultured with CFSE-stained T cells in U-bottom 96 well plates (DCs:T cells = 1:10). The cells were cultured for 72 h at 37 °C in 5% CO2 and then stained with PE-labelled anti-mouse CD4
Fig. 5. Proliferation of allogeneic CD4+ T cells induced by LMW-OPS-treated DCs. BMDCs were generated from C57BL/6 mice and treated with LMW-OPS (1 mg/ml) or LPS (1 μg/ml) for 24 h. The proliferation of CD4+ T cells was evaluated with co-cultured CFSE-labelled BALB/c mouse-derived T cells (DCs:T = 1:10) and using flow cytometry three days after the co-culture (A, B). The proliferation of T cells was determined by measuring [3H]thymidine uptake in three days after co-cultured BMDCs with allogeneic T cells (C). The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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antibody. CD4+ T cell expansion was detected by CFSE dilution using a flow cytometer. The cell culture supernatants were obtained and stored at −80 °C until IFN-γ and IL-2 determination using the ELISA kits. For T cell proliferation assays, the BMDCs from C57BL/6 mice were treated with 20 μg/ml mitomycin C for 2 h. The mitomycin C-treated BMDCs (4 × 104 cells) were added to 4 × 105 T cells in U-bottom 96 well plates (DCs:T cells = 1:10). The cells were co-cultured for 72 h at 37 °C in 5% CO2, and the allogenic T cells from BALB/c mice were pulse-labelled with [3H]thymidine (1.0 μCi/well, 5.0 Ci/mmol) for 16 h and then harvested on glass fiber filters using an automated cell harvester. The amount of [3H]thymidine incorporated was measured using a liquid scintillation counter. 2.7. Western blotting For Western blot analysis, control or LMW-OPS-treated DCs were lysed, and proteins from cell lysates were loaded onto 10% SDS-PAGE gels. Western blotting analysis was conducted by using the following primary antibodies: anti-phospho-p46 JNK/p54 JNK, anti-p46 JNK, anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-ERK1/ERK2 and anti-ERK1. The immunoblots were visualized using ECL detection reagents (Amersham). 2.8. Data analysis The results are expressed as the means and SDs of more than three independent experiments. Student's t-test was used to analyse data in
two groups. Tukey's test (SPSS) was following multiple comparisons analysed by one-way ANOVA. A p value b 5% was regarded as statistically significant.
3. Results 3.1. LMW-OPS-induced phenotypic maturation of DCs LMW-OPS increased the surface expression of MHC-II (Fig. 1A and B), CD86 (Fig. 1A and C), and CD40 (Fig. 1A and D), which are markers of DC maturation, in a dose-dependent manner in BMDCs. LMW-OPS also significantly enhanced the expression of MHC-II and CD86 in splenic DCs after intraperitoneal administration in vivo (Fig. 1E, F and G). In the in vivo experiment, no difference was observed between control mice and mice treated with LMWOPS in final body weights gains (data not shown). To exclude the possibility that LPS contamination of the LMW-OPS produced the up-regulation of these markers, we performed two experiments; one experiment used polymyxin B (PMB), which can bind to and neutralize LPS, and the other experiment used BMDCs obtained from C3H/HeJ mice, which express mutated Toll-like receptor 4 (TLR4). The LMW-OPS-induced up-regulation of MHC-II and CD86 expression was not influenced by PMB, which blocked the effects of LPS (Fig. 2A–D). LMW-OPS significantly increased MHC-II and CD86 expression on the surface of BMDCs obtained from C3H/HeJ mice (Fig. 3A–C).
Fig. 6. LMW-OPS-mediated enhancement of Th1 cytokine production induced by allogeneic responses. BMDCs were generated from C57BL/6 mice and treated with LMW-OPS (1 mg/ml) or LPS (1 μg/ml) for 24 h and then co-cultured with BALB/c mouse-derived T cells (DCs:T = 1:10). The levels of IL-2 (A), IFN-γ (B), and IL-4 (C) released during the 3 h of co-culture were measured. The values are presented as the means ± SD (n = 3). A value of *p b 0.05 is regarded as statistically significant.
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3.2. Induction of IL-12 and TNF-α in BMDCs stimulated with LMW-OPS LMW-OPS also induced cytokine production in BMDCs. IL-12 and TNF-α, which were significantly decreased in the BMDCs obtained from MyD88−/− mice, were induced by LMW-OPS in a dose-dependent manner (Fig. 4A, B, D and E). LPS did not induce the secretion of IL-12 and TNF-α in the BMDCs obtained from TLR4 mutant C3H/HeJ mice (data not shown). LMW-OPS-induced release of these cytokines was not changed in the BMDCs of C3H/HeJ mice (Fig. 4C and F), and not impacted by polymyxin B in the BMDCs of C3H/HeN mice (data not shown), indicating that TLR4 is not involved in the action of LMW-OPS. 3.3. LMW-OPS-mediated enhancement of T cell expansion and Th1 cytokine release in mixed lymphocyte reactions We investigated whether LMW-OPS-activated BMDCs could enhance T cell responses. Proliferation of T cells (Fig. 5C) and CD4+ T cells (Fig. 5A and B) induced by mixed lymphocyte reactions was enhanced in BMDCs pre-treated with LMW-OPS or LPS (Fig. 5). Allogeneic secretion of IL-2 (Fig. 6A) and IFN-γ (Fig. 6B) was also significantly increased in BMDCs pre-treated with LMW-OPS. In contrast, production of IL-4 was not affected (Fig. 6C). 3.4. Increased phosphorylation of MAPKs in BMDCs treated with LMW-OPS To understand the underlying mechanisms of the changes in BMDCs, we investigated changes in the phosphorylation levels of MAPKs, including ERK, JNK, and p38 MAPK, in BMDCs stimulated with LMWOPS. We observed that both ERK1/2 and p38 MAPK phosphorylation
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levels were significantly up-regulated at 5 and 15 min after LMW-OPS treatment (Fig. 7A–C). However, LMW-OPS had no effect on JNK phosphorylation (Fig. 7A and D). 4. Discussion Studies have shown that the level of DC maturation determines the type of DC-induced immune response. The expression levels of MHCII, CD86 and CD40 are much higher in mature DCs, which have a strong ability to initiate T cell activation. However, immune tolerance can be induced if the MHC-II and CD86 expression on the surface of DCs is decreased in the tumour microenvironment [15,16]. Previous studies have found that immunomodulators, such as plant and medicinal fungus-derived polysaccharides, induce DC maturation during tumour immunotherapy [10,11,17,18]. In our study, we found that low levels of expression of MHC-II, CD86 and CD40 in immature BMDCs were significantly up-regulated by LMW-OPS (Fig. 1–3), we also observed that MHC-II and CD86 expression in splenic DCs were enhanced by in vivo treated with LMW-OPS. Our discovery indicates that LMW-OPS can induce DC maturation. T cell-mediated immune responses, which play an important role in anti-tumour immunity, and particularly their secretion of various cytokines are largely regulated by DCs [19,20]. LMW-OPS stimulated the secretion of TNF-α and IL-12, which are involved in Th1 skewing. Furthermore, LMW-OPS treatment drastically augmented the secretion of IL-2 and IFN-γ in BMDCs during allogeneic responses. These results strongly indicate that LMW-OPS induce T-cell polarization to Th1. Current research shows that the TLR signalling pathway has a crucial function in DCs by initiating the immune response, and ligation of TLRs
Fig. 7. Activation of MAPKs by LMW-OPS. BMDCs were generated from C57BL/6 mice and treated with or without LMW-OPS (1 mg/ml) for the indicated periods. Phosphorylation of ERK (B), p38 MAPK (C), and JNK (D) was measured by immunoblot analysis. The image (A) is representative of five independent experiments. The values are presented as the means ± SD (n = 5). A value of *p b 0.05 is regarded as statistically significant.
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can lead to rapid DC activation and generate Th1-polarizing DCs [21– 26]. Studies have also shown that except for TLR3, MyD88 is a signal transducing adaptor protein that activates the TLR signalling pathway [27,28]. Therefore, we tested whether the mechanism of LMW-OPS-induced DC maturation was related to the MyD88 signalling pathway. As shown in Fig. 4B and D, LMW-OPS-induced release of IL-12 and TNF-α was significantly reduced in BMDCs obtained from the MyD88−/− mice, but not changed in the C3H/HeJ mice (Fig. 4D and F), which indicates that LMW-OPS-induced DC maturation or activation relies on TLRs/ MyD88 signalling in a TLR4-independent manner. However, the secretion of TNF-α and IL-12 was not completely eliminated in BMDCs obtained from MyD88−/− mice, suggesting that LMW-OPS can also interact with other signal transduction pathways such as the TRIF pathway [29,30]. The phosphorylation levels of ERK and P38 MAPK were up-regulated by LMW-OPS. Studies have shown that TLR3 uses TRIF adaptor molecules [29–33], such as the TLR3/TRIF/MAPK signal transduction pathway [33]. However, the secretion of IL-12 and TNF-α in immature BMDCs by polysaccharides such as β-glucan extracts is dependent on the dectin-1 pathway [34]. Therefore, we are currently further analysing the mechanism of LMW-OPS-induced DC maturation in the TLR-dependent and independent pathways. Recently, we found that oral administration of the water-ethanol extract of oysters could suppress tumour progression in tumour-bearing mice and activate natural killer cells in vitro (unpublished results). Furthermore, the enhanced effects of crude polysaccharides derived from oysters on antigen-specific Th1 immunity was also confirmed in vivo by Cheng et al. [13]. The present paper describes the purification of LMW-OPS, which are homogeneous polysaccharides, and the investigation of their effects on DC activation. The LMW-OPS-induced functional and phenotypic maturation of DCs is characterized by a robust increase in the expression of CD86, CD40 and MHC-II and the secretion of TNF-α and IL-12. This mechanism triggers the secretion of IL-2 and IFN-γ by naïve CD4+ T cells, which is a response of Th1 cells. Oysters are cultivated in many countries, and oyster dietary supplements are widely consumed. Our results suggest that LMW-OPS are active ingredients from oysters that affect dendritic cell-mediated immunity and can be utilized as a potential adjuvant to enhance the anti-tumour effects of DC based therapy. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2017.01.018. Acknowledgements This work was supported by the Shandong Provincial Natural Science Foundation, China (Grant No. ZR2014HM038). We thank Professor Satoshi Tanaka (Okayama University) for the valuable comments on this study. References [1] E. Gilboa, DC-based cancer vaccines, J. Clin. Invest. 117 (5) (2007) 1195–1203. [2] J. Banchereau, A.K. Palucka, Dendritic cells as therapeutic vaccines against cancer, Nat. Rev. Immunol. 5 (4) (2005) 296–306. [3] K. Palucka, J. Banchereau, Cancer immunotherapy via dendritic cells, Nat. Rev. Cancer 12 (4) (2012) 265–277. [4] K. Palucka, H. Ueno, J. Banchereau, Recent developments in cancer vaccines, J. Immunol. 186 (3) (2011) 1325–1331. [5] F.S. Lichtenegger, K. Mueller, B. Otte, et al., CD86 and IL-12p70 are key players for T helper 1 polarization and natural killer cell activation by Toll-like receptor-induced dendritic cells, PLoS One 7 (9) (2012), e44266. . [6] M.P. Longhi, C. Trumpfheller, J. Idoyaga, et al., Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant, J. Exp. Med. 206 (7) (2009) 1589–1602.
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