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Exopolysaccharide from Trichoderma pseudokoningii promotes maturation of murine dendritic cells
International Journal of Biological Macromolecules 92 (2016) 1155–1161
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Exopolysaccharide from Trichoderma pseudokoningii promotes maturation of murine dendritic cells Yanghui Xu a,d,1 , Jing Li a,c,1 , Jing Ju d , Bingxiang Shen c , Guochuang Chen b , Wen Qian a , Lei Zhu a , Jingbo Lu a , Chunyan Liu a , Guozheng Qin a , Guodong Wang a,∗∗ , Kaoshan Chen a,b,∗ a Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Anhui Province Key Laboratory of Active Biological Macro-molecules, School of Pharmacy, Wannan Medical College, Wuhu 241000, China b School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan 250100, China c Department of Pharmacy, Lu’an Affiliated Hospital of Anhui Medical University, Lu’an 237000, China d Department of Pharmacy, Anqing Hospital Affiliated to Anhui Medical University, Anqing 246000, China
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Article history: Received 29 April 2016 Received in revised form 18 June 2016 Accepted 20 June 2016 Available online 21 June 2016 Keywords: Exopolysaccharide Trichoderma pseudokoningii Murine dendritic cells Maturation Nuclear factor-B p38 MAPK
a b s t r a c t Dendritic cells (DCs) are the key regulators of immune responses. In this study, the effect of an exopolysaccharide (EPS) from the culture broth of Trichoderma pseudokoningii on the phenotypic and functional maturation of murine DCs and its underlying molecular mechanisms were investigated. It showed that EPS induced the morphological changes of DCs and the enhanced expression of DCs featured surface molecules CD11c, CD86, CD80 and major histocompatibility complex II (MHC-II). Flow cytometry analysis showed that the treatment with EPS could reduce FITC-dextran uptake by DCs. Sequentially, the results of ELISA indicated that EPS could increase the production of interleukin-12p70 (IL-12p70) in culture supernatant of DCs. Immunofluorescence staining and western blot analysis further revealed that EPS significantly prompted nuclear factor (NF)-B subunit p65 translocation, IB-␣ protein degradation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation. And the production of IL-12p70 was significantly decreased in condition of the inhibition of p38 or NF-B signaling pathway. These findings suggested that EPS could induce DCs maturation through both p38 MAPK and NF-B signaling pathways. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Dendritic cells (DCs), originally identified by Steinman (in 1973), are the predominant antigen-presenting cells (APCs) of the immune system, which play an important role in the initiation and regulation of the immune responses [1]. Studies have demonstrated that there functionally exist two types of DCs, which are commonly referred to as immature and mature DCs. Immature DCs, distribute throughout the body and possess potent capability of capturing and processing antigens. After antigen capture and process, immature DCs migrate to the T cell regions of lymphoid organs where they
∗ Corresponding author at: School of Life Science and National Glycoengineering Research Center, Shandong University, 27 Shanda Nanlu, Jinan 250100, China. ∗∗ Corresponding author at: Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Anhui Province Key Laboratory of Active Biological Macromolecules, School of Pharmacy, Wannan Medical College, 22 West of Wenchang Road, Wuhu 241000, China. E-mail addresses: [email protected] (G. Wang), [email protected] (K. Chen). 1 Both authors contributed equally to this work, thus share the first authorship. http://dx.doi.org/10.1016/j.ijbiomac.2016.06.064 0141-8130/© 2016 Elsevier B.V. All rights reserved.
transform into mature DCs [2–4]. DCs maturation is critical for activating antigen-specific T lymphocyte responses and which play an important role in antitumor activity [5]. However, a small number of immature DCs in tumor microenvironments cannot evoke antitumor immune responses instead of inducing tumor immune tolerance or anergy, which is a major obstacle that limits the efficiency of cancer immunotherapy [6]. Thus the maturation is critical for DCs to activate antitumor T-cell responses [7]. DCs maturation is accompanied with a series of changes including upregulation of cell surface MHC II and costimulatory molecules, loss of endocytic capacity and the release of multiple cytokines [8–10]. Various biological active substances can induce DCs maturation, such as bacterial products lipopolysaccharide, proinflammatory cytokines and polysaccharides [11–13]. Many natural polysaccharides exhibit potent immunostimulatory activity and could serve as an ideal candidate as the immunomodulators given the negligible side effects [14]. For example, a polysaccharide fraction purified from Portulaca oleracea L., is able to induce murine bone marrow derived dendritic cells maturation including upregulation the expression of CD80, CD86, CD83, and major histocompatibility
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complex class II (MHC-II) molecules, induction of interleukin (IL)12 and tumor necrosis factor-␣ [15]. Bioactive polysaccharides can trigger multiple signaling pathways via pattern recognition receptors (PRRs) on the surface of DCs, followed by inducing phenotypic and functional maturation of DCs [16,17]. Studies have indicated that the nuclear transcription factor (NF)-B which induce the expression of various gene involved in immune responses, plays an important role in DC maturation [18–20]. Another important signaling pathway involved in DC maturation is p38 MAPK signaling pathway. It regulates the production of proinflammatory molecules and other responses of the immune system [21,22]. It had been proved that exopolysaccharide (EPS) isolated from the culture broth of Trichoderma pseudokoningii inhibited the proliferation of human leukemia K562 and breast cancer MCF-7 cells by inducing cell apoptosis in vitro [23,24]. However, whether EPS could enhance the response of immune system, especially the function of DCs remains unknown. In this study, we investigated the effect of EPS on maturation of DCs and the possible molecular mechanism was also explored.
2.3. Cell culture The murine DCs 2.4 cell line were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% inactivated FBS, 100 U/ml penicillin and 100 g/ml streptomycin in a humidified 5% CO2 incubator at 37 ◦ C. 2.4. Assay for cell viability The DCs were seed in a 96-well plate in 100 l RPMI-1640 complete culture medium per well. Subsequently, cells were incubated with different concentrations of EPS (2.5–200 g/ml) for 48 h. Following each well was added by 10 l of MTT (5 mg/ml) and incubated for additional 4 h. Then the supernatant was removed carefully and 100 l of DMSO was added into each well. The 96well plate was shaken for 10 min until no particulate matter was visible. Absorbance at 570 nm was detected by a microplate reader (Bio-Rad, USA) for each well.
2. Materials and methods
2.5. Morphologic observations
2.1. Materials
The DCs were grown in a 6-well plate (bottom placed cover slides per well) in 1 ml RPMI-1640 complete culture medium per well. Then the cells were incubated with EPS (100 g/ml) or LPS (2 g/ml) for 10 h. The morphological changes were observed and photographed using an inverted microscope (Olympus, Japan).
Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum, penicillin and streptomycin were purchased from Gibco (Gaithersburg, USA). Anti-IB-␣ antibody, anti-actin antibody, anti-phosphorylated-p38 MAPK antibody, anti-p38 MAPK antibody, HRP-labeled goat anti-Rabbit IgG (H + L), HRP-labeled goat anti-Mouse IgG (H + L), inhibitors BAY 11-7082 and SB 203580 were supplied by Beyotime Institute of Biotechnology (Haimen, China). Sephadex G-75 were obtained from General Electric Healthcare Life Sciences. D301 R resin was provided by Nankai University. BCA protein assay kit was obtained from Thermo Fisher Scientific (Rockford, USA). CD11c, CD80, CD86 and MHC-II antibodies used for flow cytometry analysis were purchased from eBioscience (Santiago, USA). Lipopolysaccharide (LPS) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, USA). All other chemical reagents were Sigma grade.
2.6. Phenotype analysis DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C for 48 h. After incubation, cells were washed twice with phosphate buffer saline (PBS) and incubated with PE-conjugated anti-CD11c, APC-conjugated anti-MHC II, PE-Cyanine5-conjuagated anti-CD80 and FITC-conjugated anti-CD86 antibodies at 4 ◦ C for 30 min in the dark and then washed with PBS for several times. Cells were analyzed with a FACSVerseTM flow cytometer (Becton-Dickinson, USA) followed by FACSuite software (BD Biosciences, USA) analysing the data. 2.7. Assay for endocytic activity
2.2. Preparation of the exopolysaccharide from the culture broth of Trichoderma pseudokoningii Exopolysaccharide (EPS) from Trichoderma pseudokoningii was prepared as described previously [24]. Briefly, Trichoderma pseudokoningii was inoculated on Potato Dextrose Agar (PDA) medium and then incubated on a rotary shaker under the conditions of 180 rpm at 28 ◦ C for 10 days. The concentrated supernatant fermentation was obtained by filtration and reduced pressure evaporation. Then 3 times volume of 95% alcohol was slowly added into it. The mixture was kept at 4 ◦ C overnight and precipitate was collected by centrifugation and dissolved in distilled water. The resulting solution was decolorized with D301R type resin and deproteinated using the Sevag method [25]. The resulting polysaccharides solution was dialyzed against distilled water for 48 h and lyophilized to obtain crude polysaccharides. The crude polysaccharides were fractionated by a Sephadex G-75 column. The polysaccharide fraction forming major peak was collected and lyophilized to get a purified polysaccharide (EPS). The average molecular weight (31.9 kDa) measured by gel permeation chromatography (GPC) and component sugars of EPS determined by gas chromatography (GC) (rhamnose, xylose, fucose, mannose, glucose, and galactose in a molar ratio of 16.2: 14.4: 1: 25.8: 23.6: 48.1) were the same to our previous results [23].
To evaluate the endocytosis of DCs influenced by EPS, DCs were cultured with EPS (100 g/ml) or LPS (2 g/ml) for 24 h and then incubated with 1 mg/ml FITC-dextran (42,000 Da, Sigma, St. Louis, USA) at 37 ◦ C for 1 h. After incubation, the reaction was stopped by cold washing buffer (PBS containing 2% FBS). The stained DCs were analyzed by a FACSVerseTM flow cytometer and parallel experiment was performed at 4 ◦ C to confirm nonspecific binding of FITC-dextran. 2.8. Measurement of IL-12p70 level in DCs culture using ELISA DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C in 24-well plates. After 24 h incubation, the levels of IL-12p70 in the culture supernatant were determined by ELISA kit (R & D systems, Minneapolis, MN) according to the manufacturer’s instructions. 2.9. Immunofluorescence staining analysis of nuclear translocation of NF-B Adherent DCs were grown on cover slips in a 6-well plate and treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C for 30 min. Cells were fixed and incubated with a blocking buffer for 1 h to suppress non-specific binding and then cells were incubation with the
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2.8. Statistical analysis Analysis was performed using one-way analysis of variance (ANOVA) and all data were presented as means ± SD of at least three independent experiments. p-Value of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Effect of EPS on the viability of DCs
Fig. 1. Effect of EPS on cell viability of DCs. DCs were treated with EPS (2.5–200 g/ml) for 48 h followed by the MTT assay. The results were expressed as means ± SD (n = 6). Significant difference from the control group was designated as *P < 0.05.
Given that high concentration polysaccharides may show cytotoxic effect on cells, so we checked the non-toxic working concentration of the EPS for DCs. As shown in Fig. 1, EPS did not affect the viability of DCs under the concentrations even at 200 g/ml, which indicated that EPS possessed negligible toxicity on DCs. 3.2. Morphological changes of the DCs induce by EPS
primary NF-B p65 antibody at 4 ◦ C overnight, followed by incubation with a Cy3-conjugated secondary antibody at 25 ◦ C for another 1 h. After staining with DAPI for 5 min, the prepared samples were visualized and photographed under a fluorescent inverted microscope (Olympus, Japan). 2.10. Western blot analysis After treated with EPS (100 g/ml) or LPS (2 g/ml) at the indicated time periods. Cells were washed with ice cold PBS (3 times), followed by lysing with 100 l cell lysis buffer (1% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCl, 2 mM sodium pyrophosphate, 1 mM Phenylmethane-sulfonyl fluoride, 25 mM -glycerophosphate, 1 mM Na3 VO4 , 1 mM EDTA, 0.5 g/ml leupeptin). The obtained lysate was shaken for 20 min at 4 ◦ C, and then centrifugated at 10,000 g for 30 min to obtain the cytosolic fraction. Protein concentrations were determined by BCA protein assay kit according to the manufacturer instructions. SDS-sample buffer was added into the extracts and the mixture was incubated at 100 ◦ C for 5 min. Samples containing 20 g of protein were separated by 12% SDS-PAGE and electro-transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with TBS-0.1% Tween 20 (TBST) containing 5% skim milk at 25 ◦ C for 2 h, and then incubated with primary antibodies at 4 ◦ C overnight. Next, the membrane was washed three times with TBST followed by incubation with HRP-labeled goat anti-Rabbit IgG (H + L) or HRPlabeled goat anti-Mouse IgG (H + L) at 25 ◦ C for another 2 h. The blots were detected by an enhanced chemiluminescence detection kit (Beyotime, China).
As shown in Fig. 2, compared with the control group, the morphology of the DCs changed obviously after treated with LPS (2 g/ml) or EPS (100 g/ml) for 10 h. The morphology of DCs was approximately round in the control group, while the cells showed polygonal and fusiform morphology in LPS (2 g/ml) and EPS (100 g/ml) treatment groups. Morphological change of DCs could be treated as a reference for distinguishing between mature and immature DCs [26]. The result showed that EPS may induce maturation of DCs. 3.3. Upreguation of expression of the featured surface molecules on DCs treated with EPS High expression of major histocompatibility complex class II and costimulatory molecules are known as maturation markers of DCs. Through which mature DCs can sensitize CD8+ T cells which is essential for DCs to activate antitumor T-cell responses [27]. When treated with EPS (100 g/ml) or LPS (2 g/ml) for 48 h, the immature DCs transformed into mature DCs as reflected by upregulation of expression of featured surface molecules of MHC-II, CD86, CD80 and CD11c. As shown in Fig. 3, for MHC-II, EPS (100 g/ml) or LPS (2 g/ml) increased the proportion of fluorescence positive cells from 25.40% in control group to 53.66% and 68.18% in treated groups respectively. Similarly, the proportion of fluorescence positive cells increased from 25.03%, 22.25% and 12.54% in control group to 43.49%, 64.21% and 35.31% in EPS treatment group for CD86, CD80 and CD11c respectively. The results indicated that EPS could induce phenotypic maturation of DCs. It consistent with the above morphological study.
Fig. 2. The effect of EPS on morphological changes of DCs. DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) for 10 h. The morphological changes were observed and photographed under a phase contrast microscope (Olympus, Japan). A displayed the negative control group, B displayed LPS (2 g/ml) positive control group, C displayed EPS (100 g/ml) treatment group.
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Fig. 3. EPS upregulated the expression of featured surface molecules of DCs. DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C for 48 h, the changes of the expression of featured surface molecules were determined by a FACSVerseTM flow cytometer (Becton-Dickinson, USA). Data represents the percentages of fluorescence positive cells. Significant difference from the control group was designated as *P < 0.05 and **P < 0.01.
3.4. EPS inhibited the FITC-dextran uptake of DCs The endocytic capacity of immature DCs, mediated by mannose receptor, is basis for capturing and processing antigens. However, when transform into mature DCs, they will lose this capacity [28]. Studies have revealed that the endocytic capacity of DCs is inhibited by LPS during maturation process. Here we studied whether EPS affected the FITC-dextran uptake of DCs. As shown in Fig. 4, either in EPS (100 g/ml) or LPS (2 g/ml) treatment group, the percentage of FITC-dextran positive DC2.4 cells were 27.38% and 14.19%
respectively, which were significantly lower than the RPMI-1640 control group (40.19%). FITC-dextran uptake of DCs was inhibited at 4 ◦ C, suggesting that DCs endocytosis was a specific active action. Loss of endocytic capacity further suggested that EPS could induce functional maturation of DCs. 3.5. Effect of EPS on the secretion of IL-12p70 in DCs Enhanced production of IL-12p70 by DCs not only induces DCs expansion in autocrine and paracrine manner, but also is critical
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Fig. 4. The effect of EPS on endocytotic activity of DCs. DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) for 24 h, the uptake of dextran by DCs was determined by a FACSVerseTM flow cytometer (Becton-Dickinson, USA). Parallel experiments were performed at 4 ◦ C to confirm that uptake of dextran by DCs was a nonspecific binding process. Data represents the percentages of fluorescence positive cells. Significant difference from the RPMI-1640 (37 ◦ C) group was designated as *P < 0.05 and **P < 0.01.
Fig. 5. The secretion of IL-12p70 in culture supernatant of DCs induced by EPS. Cells were incubated with EPS (100 g/ml) or LPS (2 g/ml) for 24 h. The level of IL-12p70 in culture supernatant of DCs was measured by ELISA. These results were expressed as mean ± SD (n = 3). Significant difference from the control group was designated as *P < 0.05 and **P < 0.01.
for CD4+T cell activation which in turn secretes other cytokines such as TNF-and IFN-␥[26]. In this case, IL-12p70 is an important cytokine initiating the chain of immune responses. As shown in Fig. 5, EPS significantly increased the secretion of IL-12p70 in DCs at 100 g/ml. The production of IL-12p70 was increased more than 2 times compared with the control group. It suggested that EPS could induce functional maturation of DCs through secretion of IL-12p70 which may creative an immune network keeping the balance of the body. 3.6. Effect of EPS on nuclear translocation of NF-B in DCs NF-B is a widely existed transcription factor that plays a critical role in DCs maturation via regulation of a variety of genes related to immune responses [29]. In resting state, NF-B binds to its inhibitor (IB-␣) formed an inactive complex which inhibits NFB nuclear translocation from the cytoplasm to the nucleus. When stimulated by inflammatory factors, IB-␣ is phosphorylated and degraded which lead to the release of NF-B, and then the p65 subunit of NF-B nuclear translocation happens [30,31]. Here we used immunofluorescence staining to study the NF-B nuclear translocation. As shown in Fig. 6, p65 subunit of NF-B showed weak red fluorescence and scattered distribution in control group. When the
Fig. 6. EPS induced the subunit p65 of NF-B nuclear translocation occurring. Cells were treated with EPS (100 g/ml) or LPS (2 g/ml) for 30 min. And then the cells were incubated with p65 antibody and Cy3 fluorescein-conjugated secondary antibody, and DAPI was used to stain the nuclei. The pictures were photographed by a fluorescent inverted microscope (Olympus, Japan). The red fluorescence area (representative of the area that contains p65) and blue fluorescence area (representative of the nucleus that DAPI stained) pictures were merged to create a purple fluorescence in areas of colocalization. A displayed the control group, B displayed LPS (2 g/ml) positive control group, C displayed EPS (100 g/ml) treatment group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) for 30 min, the red fluorescence of p65 subunit became bright and showed concentrated distribution. The merged picture showed that a lot of p65 subunit translocated in the nucleus of DCs, while only a small portion of p65 subunit in the nucleus of DCs in the control group. When encountered stimulation, IB-␣ is phosphorylated and then degradated via ubiquitination. The free NF-B dimer is translocated to the nucleus leading to the transcription of target genes. As show in Fig. 7A. In LPS treatment group, IB-␣ reduced gradually within 5–60 min, but in the group treated with EPS, IB-␣ content didn’t show the same trend. This result indicated that the intracellular signal transduction of DCs induced by EPS and LPS seemed different. All these results demonstrated that EPS could induce p65 subunit of NF-B nuclear translocation in DCs.
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Fig. 7. EPS induced degradation of IB-␣ and phosphorylation of p38 MAPK in DCs. DCs were incubated with EPS (100 g/ml) or LPS (2 g/ml) for the indicated time periods, And then the cell lysate was collected and the level of the degradation of IB-␣ and phosphorylation of p38 MAPK were determined by western blot. The anti-actin antibody and anti-p38 MAPK antibody were used as the control blots, respectively.
Fig. 8. Effects of specific enzyme inhibitors on the secretion of IL-12p70 induced by EPS in DCs. DCs were pre-incubated with BAY 11-7082 (20 M) and SB203580 (30 M) for 30 min, followed by EPS (100 g/ml) incubation for 24 h. The concentration of IL-12p70 was measured by ELISA. Significant difference from the positive control group was designated as *P < 0.05 and **P < 0.01.
3.7. EPS induced p38 MAPK phosporylation in DCs The p38 MAPK signaling pathway also was involved in inducing DCs maturation, especially regulating key surface molecules expression and the secretion of cytokines [32]. As shown in Fig. 7B, both EPS (100 g/ml) and LPS (2 g/ml) significantly increased p38 MAPK phosphorylation and the blots of phosphorylated p38 MAPK were quite obvious at 30 min. The result indicated that EPS could activate p38 MAPK pathways in DCs. 3.8. The involvement of NF-B and p38 MAPK pathways in the secretion of IL-12p70 induced by EPS in DCs In the above experiment we found that nuclear NF-B and p38 MAPK signing pathways could be activated by EPS. Herein we investigated whether NF-B and p38 MAPK signing pathways were involved in the secretion of IL-12p70 in DCs treated with EPS. We blocked both NF-B and p38 MAPK pathways by their specific inhibitors BAY11-7082 and SB203580. As shown in Fig. 8, EPS induced IL-12p70 secretion was significantly blocked by BAY117082 and SB203580. However, the secretion of IL-12p70 were not completely blocked by the inhibitors compared with the control group which indicated other signaling pathways may be involved in the process of EPS inducing functional maturation of DCs. It is well known that extracellular signal-regulated kinase (ERK), Jun Nterminal kinase (JNK) and p38 MAPK constitute mitogen-activated protein kinases (MAPKs) [33]. In this study, we revealed p38 was involved in the process of EPS inducing functional maturation of DCs. Whether ERK and JNK contribute to DCs maturation needs further study. However, as reported, other transcription factors such as AP-1 and STAT participated in the immune response related genes [34]. Hence, we would like to investigate the roles of other transcription factors in the near future. 4. Conclusions Our study demonstrated that the extracellular polysaccharide (EPS) isolated from the culture broth of Trichoderma pseudokoningii induced the phenotypic and functional maturation of murine DCs. NF-B and p38 MAPK pathways played important roles in this maturation process. These findings provide a theoretical support for developing EPS as an immunotherapy drug. Future studies should
be performed to clarify the detailed molecular mechanisms of EPS inducing maturation of DCs and the immunoregulatory effect of EPS in vivo. Acknowledgements This study was supported by Anhui Provincial Natural Science Foundation (no. 1408085MH197 & 1508085MH191), the Key Project of Science and Technology of Shandong Province (no. 2015ZDJS04002), the Natural Science Foundation of Education Department of Anhui Province (no. KJ2015A199 and KJ2016A723), the Key Program for the Excellent Young Talents Foundation of the Education Department of Anhui Province (no. 2013SQRL054ZD), and the Performance Project of Key Laboratory of Anhui Province (no. 1606C08226). References [1] R.M. Steinman, Dendritic cells: understanding immunogenicity, Eur. J. Immunol. 37 (2007) 53–60. [2] C. Coquerelle, M. Moser, DC subsets in positive and negative regulation of immunity, Immunol. Rev. 234 (2010) 317–334. [3] R.M. Steinman, Some interfaces of dendritic cell biology, APMIS 111 (2003) 675–697. [4] D. Huang, S. Nie, L. Jiang, M. Xie, A novel polysaccharide from the seeds of Plantago asiatica L. induces dendritic cells maturation through toll-like receptor 4, Int. Immunopharmacol. 18 (2014) 236–243. [5] J.Y. Kim, J.M. Yoon Yd Fau- Ahn, J.S. Ahn Jm Fau- Kang, S.-K. Kang Js Fau- Park, K. Park Sk Fau- Lee, K.B. Lee, K. Fau- Song, H.M. Song Kb Fau- Kim, S.-B. Kim Hm Fau- Han, S.B. Han, Angelan isolated from Angelica gigas Nakai induces dendritic cell maturation through toll-like receptor 4, Int. Immunopharmacol. 7 (2007) 78–87. [6] F.A. Harding, J.A. McArthur Jg Fau- Gross, D.H. Gross Ja Fau- Raulet, J.P. Raulet Dh Fau- Allison, J.P. Allison, CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones, Nature 356 (1992) 607–609. [7] G. Angelini, M. Gardella, S. Fau- Ardy, M.R. Ardy, M. Fau- Ciriolo, G. Ciriolo Mr Fau- Filomeni, G. Filomeni, G. Fau- Di Trapani, F. Di Trapani, G. Fau- Clarke, R. Clarke, F. Fau- Sitia, A. Sitia, R. Fau- Rubartelli, A. Rubartelli, Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 1491–1496. [8] A. Chow, W. Toomre, D. Fau- Garrett, I. Garrett, W. Fau- Mellman, I. Mellman, Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane, Nature 418 (2002) 988–994. [9] C. Casals, M. Barrachina, M. Fau- Serra, J. Serra, M. Fau- Lloberas, A. Lloberas, J. Fau- Celada, A. Celada, Lipopolysaccharide up-regulates MHC class II
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Exopolysaccharide from Trichoderma pseudokoningii promotes maturation of murine dendritic cells Yanghui Xu a,d,1 , Jing Li a,c,1 , Jing Ju d , Bingxiang Shen c , Guochuang Chen b , Wen Qian a , Lei Zhu a , Jingbo Lu a , Chunyan Liu a , Guozheng Qin a , Guodong Wang a,∗∗ , Kaoshan Chen a,b,∗ a Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Anhui Province Key Laboratory of Active Biological Macro-molecules, School of Pharmacy, Wannan Medical College, Wuhu 241000, China b School of Life Science and National Glycoengineering Research Center, Shandong University, Jinan 250100, China c Department of Pharmacy, Lu’an Affiliated Hospital of Anhui Medical University, Lu’an 237000, China d Department of Pharmacy, Anqing Hospital Affiliated to Anhui Medical University, Anqing 246000, China
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Article history: Received 29 April 2016 Received in revised form 18 June 2016 Accepted 20 June 2016 Available online 21 June 2016 Keywords: Exopolysaccharide Trichoderma pseudokoningii Murine dendritic cells Maturation Nuclear factor-B p38 MAPK
a b s t r a c t Dendritic cells (DCs) are the key regulators of immune responses. In this study, the effect of an exopolysaccharide (EPS) from the culture broth of Trichoderma pseudokoningii on the phenotypic and functional maturation of murine DCs and its underlying molecular mechanisms were investigated. It showed that EPS induced the morphological changes of DCs and the enhanced expression of DCs featured surface molecules CD11c, CD86, CD80 and major histocompatibility complex II (MHC-II). Flow cytometry analysis showed that the treatment with EPS could reduce FITC-dextran uptake by DCs. Sequentially, the results of ELISA indicated that EPS could increase the production of interleukin-12p70 (IL-12p70) in culture supernatant of DCs. Immunofluorescence staining and western blot analysis further revealed that EPS significantly prompted nuclear factor (NF)-B subunit p65 translocation, IB-␣ protein degradation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation. And the production of IL-12p70 was significantly decreased in condition of the inhibition of p38 or NF-B signaling pathway. These findings suggested that EPS could induce DCs maturation through both p38 MAPK and NF-B signaling pathways. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Dendritic cells (DCs), originally identified by Steinman (in 1973), are the predominant antigen-presenting cells (APCs) of the immune system, which play an important role in the initiation and regulation of the immune responses [1]. Studies have demonstrated that there functionally exist two types of DCs, which are commonly referred to as immature and mature DCs. Immature DCs, distribute throughout the body and possess potent capability of capturing and processing antigens. After antigen capture and process, immature DCs migrate to the T cell regions of lymphoid organs where they
∗ Corresponding author at: School of Life Science and National Glycoengineering Research Center, Shandong University, 27 Shanda Nanlu, Jinan 250100, China. ∗∗ Corresponding author at: Anhui Provincial Engineering Research Center for Polysaccharide Drugs, Anhui Province Key Laboratory of Active Biological Macromolecules, School of Pharmacy, Wannan Medical College, 22 West of Wenchang Road, Wuhu 241000, China. E-mail addresses: [email protected] (G. Wang), [email protected] (K. Chen). 1 Both authors contributed equally to this work, thus share the first authorship. http://dx.doi.org/10.1016/j.ijbiomac.2016.06.064 0141-8130/© 2016 Elsevier B.V. All rights reserved.
transform into mature DCs [2–4]. DCs maturation is critical for activating antigen-specific T lymphocyte responses and which play an important role in antitumor activity [5]. However, a small number of immature DCs in tumor microenvironments cannot evoke antitumor immune responses instead of inducing tumor immune tolerance or anergy, which is a major obstacle that limits the efficiency of cancer immunotherapy [6]. Thus the maturation is critical for DCs to activate antitumor T-cell responses [7]. DCs maturation is accompanied with a series of changes including upregulation of cell surface MHC II and costimulatory molecules, loss of endocytic capacity and the release of multiple cytokines [8–10]. Various biological active substances can induce DCs maturation, such as bacterial products lipopolysaccharide, proinflammatory cytokines and polysaccharides [11–13]. Many natural polysaccharides exhibit potent immunostimulatory activity and could serve as an ideal candidate as the immunomodulators given the negligible side effects [14]. For example, a polysaccharide fraction purified from Portulaca oleracea L., is able to induce murine bone marrow derived dendritic cells maturation including upregulation the expression of CD80, CD86, CD83, and major histocompatibility
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complex class II (MHC-II) molecules, induction of interleukin (IL)12 and tumor necrosis factor-␣ [15]. Bioactive polysaccharides can trigger multiple signaling pathways via pattern recognition receptors (PRRs) on the surface of DCs, followed by inducing phenotypic and functional maturation of DCs [16,17]. Studies have indicated that the nuclear transcription factor (NF)-B which induce the expression of various gene involved in immune responses, plays an important role in DC maturation [18–20]. Another important signaling pathway involved in DC maturation is p38 MAPK signaling pathway. It regulates the production of proinflammatory molecules and other responses of the immune system [21,22]. It had been proved that exopolysaccharide (EPS) isolated from the culture broth of Trichoderma pseudokoningii inhibited the proliferation of human leukemia K562 and breast cancer MCF-7 cells by inducing cell apoptosis in vitro [23,24]. However, whether EPS could enhance the response of immune system, especially the function of DCs remains unknown. In this study, we investigated the effect of EPS on maturation of DCs and the possible molecular mechanism was also explored.
2.3. Cell culture The murine DCs 2.4 cell line were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% inactivated FBS, 100 U/ml penicillin and 100 g/ml streptomycin in a humidified 5% CO2 incubator at 37 ◦ C. 2.4. Assay for cell viability The DCs were seed in a 96-well plate in 100 l RPMI-1640 complete culture medium per well. Subsequently, cells were incubated with different concentrations of EPS (2.5–200 g/ml) for 48 h. Following each well was added by 10 l of MTT (5 mg/ml) and incubated for additional 4 h. Then the supernatant was removed carefully and 100 l of DMSO was added into each well. The 96well plate was shaken for 10 min until no particulate matter was visible. Absorbance at 570 nm was detected by a microplate reader (Bio-Rad, USA) for each well.
2. Materials and methods
2.5. Morphologic observations
2.1. Materials
The DCs were grown in a 6-well plate (bottom placed cover slides per well) in 1 ml RPMI-1640 complete culture medium per well. Then the cells were incubated with EPS (100 g/ml) or LPS (2 g/ml) for 10 h. The morphological changes were observed and photographed using an inverted microscope (Olympus, Japan).
Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum, penicillin and streptomycin were purchased from Gibco (Gaithersburg, USA). Anti-IB-␣ antibody, anti-actin antibody, anti-phosphorylated-p38 MAPK antibody, anti-p38 MAPK antibody, HRP-labeled goat anti-Rabbit IgG (H + L), HRP-labeled goat anti-Mouse IgG (H + L), inhibitors BAY 11-7082 and SB 203580 were supplied by Beyotime Institute of Biotechnology (Haimen, China). Sephadex G-75 were obtained from General Electric Healthcare Life Sciences. D301 R resin was provided by Nankai University. BCA protein assay kit was obtained from Thermo Fisher Scientific (Rockford, USA). CD11c, CD80, CD86 and MHC-II antibodies used for flow cytometry analysis were purchased from eBioscience (Santiago, USA). Lipopolysaccharide (LPS) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, USA). All other chemical reagents were Sigma grade.
2.6. Phenotype analysis DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C for 48 h. After incubation, cells were washed twice with phosphate buffer saline (PBS) and incubated with PE-conjugated anti-CD11c, APC-conjugated anti-MHC II, PE-Cyanine5-conjuagated anti-CD80 and FITC-conjugated anti-CD86 antibodies at 4 ◦ C for 30 min in the dark and then washed with PBS for several times. Cells were analyzed with a FACSVerseTM flow cytometer (Becton-Dickinson, USA) followed by FACSuite software (BD Biosciences, USA) analysing the data. 2.7. Assay for endocytic activity
2.2. Preparation of the exopolysaccharide from the culture broth of Trichoderma pseudokoningii Exopolysaccharide (EPS) from Trichoderma pseudokoningii was prepared as described previously [24]. Briefly, Trichoderma pseudokoningii was inoculated on Potato Dextrose Agar (PDA) medium and then incubated on a rotary shaker under the conditions of 180 rpm at 28 ◦ C for 10 days. The concentrated supernatant fermentation was obtained by filtration and reduced pressure evaporation. Then 3 times volume of 95% alcohol was slowly added into it. The mixture was kept at 4 ◦ C overnight and precipitate was collected by centrifugation and dissolved in distilled water. The resulting solution was decolorized with D301R type resin and deproteinated using the Sevag method [25]. The resulting polysaccharides solution was dialyzed against distilled water for 48 h and lyophilized to obtain crude polysaccharides. The crude polysaccharides were fractionated by a Sephadex G-75 column. The polysaccharide fraction forming major peak was collected and lyophilized to get a purified polysaccharide (EPS). The average molecular weight (31.9 kDa) measured by gel permeation chromatography (GPC) and component sugars of EPS determined by gas chromatography (GC) (rhamnose, xylose, fucose, mannose, glucose, and galactose in a molar ratio of 16.2: 14.4: 1: 25.8: 23.6: 48.1) were the same to our previous results [23].
To evaluate the endocytosis of DCs influenced by EPS, DCs were cultured with EPS (100 g/ml) or LPS (2 g/ml) for 24 h and then incubated with 1 mg/ml FITC-dextran (42,000 Da, Sigma, St. Louis, USA) at 37 ◦ C for 1 h. After incubation, the reaction was stopped by cold washing buffer (PBS containing 2% FBS). The stained DCs were analyzed by a FACSVerseTM flow cytometer and parallel experiment was performed at 4 ◦ C to confirm nonspecific binding of FITC-dextran. 2.8. Measurement of IL-12p70 level in DCs culture using ELISA DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C in 24-well plates. After 24 h incubation, the levels of IL-12p70 in the culture supernatant were determined by ELISA kit (R & D systems, Minneapolis, MN) according to the manufacturer’s instructions. 2.9. Immunofluorescence staining analysis of nuclear translocation of NF-B Adherent DCs were grown on cover slips in a 6-well plate and treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C for 30 min. Cells were fixed and incubated with a blocking buffer for 1 h to suppress non-specific binding and then cells were incubation with the
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2.8. Statistical analysis Analysis was performed using one-way analysis of variance (ANOVA) and all data were presented as means ± SD of at least three independent experiments. p-Value of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Effect of EPS on the viability of DCs
Fig. 1. Effect of EPS on cell viability of DCs. DCs were treated with EPS (2.5–200 g/ml) for 48 h followed by the MTT assay. The results were expressed as means ± SD (n = 6). Significant difference from the control group was designated as *P < 0.05.
Given that high concentration polysaccharides may show cytotoxic effect on cells, so we checked the non-toxic working concentration of the EPS for DCs. As shown in Fig. 1, EPS did not affect the viability of DCs under the concentrations even at 200 g/ml, which indicated that EPS possessed negligible toxicity on DCs. 3.2. Morphological changes of the DCs induce by EPS
primary NF-B p65 antibody at 4 ◦ C overnight, followed by incubation with a Cy3-conjugated secondary antibody at 25 ◦ C for another 1 h. After staining with DAPI for 5 min, the prepared samples were visualized and photographed under a fluorescent inverted microscope (Olympus, Japan). 2.10. Western blot analysis After treated with EPS (100 g/ml) or LPS (2 g/ml) at the indicated time periods. Cells were washed with ice cold PBS (3 times), followed by lysing with 100 l cell lysis buffer (1% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCl, 2 mM sodium pyrophosphate, 1 mM Phenylmethane-sulfonyl fluoride, 25 mM -glycerophosphate, 1 mM Na3 VO4 , 1 mM EDTA, 0.5 g/ml leupeptin). The obtained lysate was shaken for 20 min at 4 ◦ C, and then centrifugated at 10,000 g for 30 min to obtain the cytosolic fraction. Protein concentrations were determined by BCA protein assay kit according to the manufacturer instructions. SDS-sample buffer was added into the extracts and the mixture was incubated at 100 ◦ C for 5 min. Samples containing 20 g of protein were separated by 12% SDS-PAGE and electro-transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with TBS-0.1% Tween 20 (TBST) containing 5% skim milk at 25 ◦ C for 2 h, and then incubated with primary antibodies at 4 ◦ C overnight. Next, the membrane was washed three times with TBST followed by incubation with HRP-labeled goat anti-Rabbit IgG (H + L) or HRPlabeled goat anti-Mouse IgG (H + L) at 25 ◦ C for another 2 h. The blots were detected by an enhanced chemiluminescence detection kit (Beyotime, China).
As shown in Fig. 2, compared with the control group, the morphology of the DCs changed obviously after treated with LPS (2 g/ml) or EPS (100 g/ml) for 10 h. The morphology of DCs was approximately round in the control group, while the cells showed polygonal and fusiform morphology in LPS (2 g/ml) and EPS (100 g/ml) treatment groups. Morphological change of DCs could be treated as a reference for distinguishing between mature and immature DCs [26]. The result showed that EPS may induce maturation of DCs. 3.3. Upreguation of expression of the featured surface molecules on DCs treated with EPS High expression of major histocompatibility complex class II and costimulatory molecules are known as maturation markers of DCs. Through which mature DCs can sensitize CD8+ T cells which is essential for DCs to activate antitumor T-cell responses [27]. When treated with EPS (100 g/ml) or LPS (2 g/ml) for 48 h, the immature DCs transformed into mature DCs as reflected by upregulation of expression of featured surface molecules of MHC-II, CD86, CD80 and CD11c. As shown in Fig. 3, for MHC-II, EPS (100 g/ml) or LPS (2 g/ml) increased the proportion of fluorescence positive cells from 25.40% in control group to 53.66% and 68.18% in treated groups respectively. Similarly, the proportion of fluorescence positive cells increased from 25.03%, 22.25% and 12.54% in control group to 43.49%, 64.21% and 35.31% in EPS treatment group for CD86, CD80 and CD11c respectively. The results indicated that EPS could induce phenotypic maturation of DCs. It consistent with the above morphological study.
Fig. 2. The effect of EPS on morphological changes of DCs. DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) for 10 h. The morphological changes were observed and photographed under a phase contrast microscope (Olympus, Japan). A displayed the negative control group, B displayed LPS (2 g/ml) positive control group, C displayed EPS (100 g/ml) treatment group.
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Fig. 3. EPS upregulated the expression of featured surface molecules of DCs. DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) at 37 ◦ C for 48 h, the changes of the expression of featured surface molecules were determined by a FACSVerseTM flow cytometer (Becton-Dickinson, USA). Data represents the percentages of fluorescence positive cells. Significant difference from the control group was designated as *P < 0.05 and **P < 0.01.
3.4. EPS inhibited the FITC-dextran uptake of DCs The endocytic capacity of immature DCs, mediated by mannose receptor, is basis for capturing and processing antigens. However, when transform into mature DCs, they will lose this capacity [28]. Studies have revealed that the endocytic capacity of DCs is inhibited by LPS during maturation process. Here we studied whether EPS affected the FITC-dextran uptake of DCs. As shown in Fig. 4, either in EPS (100 g/ml) or LPS (2 g/ml) treatment group, the percentage of FITC-dextran positive DC2.4 cells were 27.38% and 14.19%
respectively, which were significantly lower than the RPMI-1640 control group (40.19%). FITC-dextran uptake of DCs was inhibited at 4 ◦ C, suggesting that DCs endocytosis was a specific active action. Loss of endocytic capacity further suggested that EPS could induce functional maturation of DCs. 3.5. Effect of EPS on the secretion of IL-12p70 in DCs Enhanced production of IL-12p70 by DCs not only induces DCs expansion in autocrine and paracrine manner, but also is critical
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Fig. 4. The effect of EPS on endocytotic activity of DCs. DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) for 24 h, the uptake of dextran by DCs was determined by a FACSVerseTM flow cytometer (Becton-Dickinson, USA). Parallel experiments were performed at 4 ◦ C to confirm that uptake of dextran by DCs was a nonspecific binding process. Data represents the percentages of fluorescence positive cells. Significant difference from the RPMI-1640 (37 ◦ C) group was designated as *P < 0.05 and **P < 0.01.
Fig. 5. The secretion of IL-12p70 in culture supernatant of DCs induced by EPS. Cells were incubated with EPS (100 g/ml) or LPS (2 g/ml) for 24 h. The level of IL-12p70 in culture supernatant of DCs was measured by ELISA. These results were expressed as mean ± SD (n = 3). Significant difference from the control group was designated as *P < 0.05 and **P < 0.01.
for CD4+T cell activation which in turn secretes other cytokines such as TNF-and IFN-␥[26]. In this case, IL-12p70 is an important cytokine initiating the chain of immune responses. As shown in Fig. 5, EPS significantly increased the secretion of IL-12p70 in DCs at 100 g/ml. The production of IL-12p70 was increased more than 2 times compared with the control group. It suggested that EPS could induce functional maturation of DCs through secretion of IL-12p70 which may creative an immune network keeping the balance of the body. 3.6. Effect of EPS on nuclear translocation of NF-B in DCs NF-B is a widely existed transcription factor that plays a critical role in DCs maturation via regulation of a variety of genes related to immune responses [29]. In resting state, NF-B binds to its inhibitor (IB-␣) formed an inactive complex which inhibits NFB nuclear translocation from the cytoplasm to the nucleus. When stimulated by inflammatory factors, IB-␣ is phosphorylated and degraded which lead to the release of NF-B, and then the p65 subunit of NF-B nuclear translocation happens [30,31]. Here we used immunofluorescence staining to study the NF-B nuclear translocation. As shown in Fig. 6, p65 subunit of NF-B showed weak red fluorescence and scattered distribution in control group. When the
Fig. 6. EPS induced the subunit p65 of NF-B nuclear translocation occurring. Cells were treated with EPS (100 g/ml) or LPS (2 g/ml) for 30 min. And then the cells were incubated with p65 antibody and Cy3 fluorescein-conjugated secondary antibody, and DAPI was used to stain the nuclei. The pictures were photographed by a fluorescent inverted microscope (Olympus, Japan). The red fluorescence area (representative of the area that contains p65) and blue fluorescence area (representative of the nucleus that DAPI stained) pictures were merged to create a purple fluorescence in areas of colocalization. A displayed the control group, B displayed LPS (2 g/ml) positive control group, C displayed EPS (100 g/ml) treatment group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
DCs were treated with EPS (100 g/ml) or LPS (2 g/ml) for 30 min, the red fluorescence of p65 subunit became bright and showed concentrated distribution. The merged picture showed that a lot of p65 subunit translocated in the nucleus of DCs, while only a small portion of p65 subunit in the nucleus of DCs in the control group. When encountered stimulation, IB-␣ is phosphorylated and then degradated via ubiquitination. The free NF-B dimer is translocated to the nucleus leading to the transcription of target genes. As show in Fig. 7A. In LPS treatment group, IB-␣ reduced gradually within 5–60 min, but in the group treated with EPS, IB-␣ content didn’t show the same trend. This result indicated that the intracellular signal transduction of DCs induced by EPS and LPS seemed different. All these results demonstrated that EPS could induce p65 subunit of NF-B nuclear translocation in DCs.
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Fig. 7. EPS induced degradation of IB-␣ and phosphorylation of p38 MAPK in DCs. DCs were incubated with EPS (100 g/ml) or LPS (2 g/ml) for the indicated time periods, And then the cell lysate was collected and the level of the degradation of IB-␣ and phosphorylation of p38 MAPK were determined by western blot. The anti-actin antibody and anti-p38 MAPK antibody were used as the control blots, respectively.
Fig. 8. Effects of specific enzyme inhibitors on the secretion of IL-12p70 induced by EPS in DCs. DCs were pre-incubated with BAY 11-7082 (20 M) and SB203580 (30 M) for 30 min, followed by EPS (100 g/ml) incubation for 24 h. The concentration of IL-12p70 was measured by ELISA. Significant difference from the positive control group was designated as *P < 0.05 and **P < 0.01.
3.7. EPS induced p38 MAPK phosporylation in DCs The p38 MAPK signaling pathway also was involved in inducing DCs maturation, especially regulating key surface molecules expression and the secretion of cytokines [32]. As shown in Fig. 7B, both EPS (100 g/ml) and LPS (2 g/ml) significantly increased p38 MAPK phosphorylation and the blots of phosphorylated p38 MAPK were quite obvious at 30 min. The result indicated that EPS could activate p38 MAPK pathways in DCs. 3.8. The involvement of NF-B and p38 MAPK pathways in the secretion of IL-12p70 induced by EPS in DCs In the above experiment we found that nuclear NF-B and p38 MAPK signing pathways could be activated by EPS. Herein we investigated whether NF-B and p38 MAPK signing pathways were involved in the secretion of IL-12p70 in DCs treated with EPS. We blocked both NF-B and p38 MAPK pathways by their specific inhibitors BAY11-7082 and SB203580. As shown in Fig. 8, EPS induced IL-12p70 secretion was significantly blocked by BAY117082 and SB203580. However, the secretion of IL-12p70 were not completely blocked by the inhibitors compared with the control group which indicated other signaling pathways may be involved in the process of EPS inducing functional maturation of DCs. It is well known that extracellular signal-regulated kinase (ERK), Jun Nterminal kinase (JNK) and p38 MAPK constitute mitogen-activated protein kinases (MAPKs) [33]. In this study, we revealed p38 was involved in the process of EPS inducing functional maturation of DCs. Whether ERK and JNK contribute to DCs maturation needs further study. However, as reported, other transcription factors such as AP-1 and STAT participated in the immune response related genes [34]. Hence, we would like to investigate the roles of other transcription factors in the near future. 4. Conclusions Our study demonstrated that the extracellular polysaccharide (EPS) isolated from the culture broth of Trichoderma pseudokoningii induced the phenotypic and functional maturation of murine DCs. NF-B and p38 MAPK pathways played important roles in this maturation process. These findings provide a theoretical support for developing EPS as an immunotherapy drug. Future studies should
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