Ginsenoside Rg1 Activates Dendritic Cells and Acts as a Vaccine Adjuvant Inducing Protective Cellular Responses Against Lymphomas

Abstract

Ginsenoside, a natural triterpenoid saponin, exhibits immunomodulatory and anticancer activities. In the present study, we demonstrated that ginsenoside Rg1 induced secretion of cytokines, including interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and IL-1β, and chemokines such as IL-8 and IP-10 in a dose-dependent manner by human peripheral blood mononuclear cell (PBMC)-derived dendritic cells. Rg1 stimulated the expression of the surface molecules CD83, CD80, and human leukocyte antigen – antigen D related (HLA-DR) and decreased the expression of CD14. In in vivo experiments, C57BL/6 mice were divided into four groups, immunized with ovalbumin (OVA), OVA plus Rg1, Rg1, and phosphate-buffered saline (PBS), respectively. Splenocytes from C57BL/6 mice immunized with OVA plus Rg1 produced more antigen-specific splenocyte proliferation activity. The level of IFN-γ and IL-4 in the splenocytes was also upregulated when in vitro stimulated with OVA_257–264 or OVA. After in vivo injection of tumor-forming E.G7-OVA cells, the survival rate of mice immunized intraperitoneally in OVA plus Rg1 immunized mice was higher than that in OVA immunized mice or PBS immunized mice. Thus, Rg1 induced a potent vaccine adjuvant effect and elicited antitumor immunity that polarized a Th1 type immune response. Rg1 could have potential as a prophylactic vaccine adjuvant to control lymphomas.

Introduction

Lymphoma is a malignant tumor in the body's immune system. There are mainly two types of lymphoma, including Hodgkin lymphoma and non-Hodgkin lymphoma (NHL) (Witzig et al., 2015; Baues et al., 2017). Lymphoma is usually grown within the tissues and organs in the form of solid tumors rich in lymphoid tissues. Lymph nodes, tonsils, spleen, and bone marrow are the most vulnerable parts of lymphomas. The common treatment for lymphomas is chemotherapy, radiation therapy, stem cell transplantation/bone marrow transplantation, etc. (Ng, 2011; Crump et al., 2015). The current therapy of high-grade lymphoma is effective; however, many patients develop drug resistance and relapse. Thus, the novel therapeutics are still needed to kill the residual cancer cells and to restore immune surveillance for maintenance of human health (Contag et al., 2010).

Preventive cancer vaccine or therapeutic cancer vaccine is a promising novel strategy to prevent or cure human cancers. For instance, CIMAvax-EGF is a therapeutic cancer vaccine for nonsmall-cell lung cancer (Cheng and Kananathan, 2012; Crombet Ramos et al., 2015; Saavedra and Crombet, 2017). BiovaxID is a patient-specific therapeutic cancer vaccine, which is used together with granulocyte-macrophage colony-stimulating factor in a phase II clinical trial for follicular lymphoma. It induces tumor-specific cellular and humoral immunity and is associated with prolonged disease-free survival (Lee et al., 2007). Dendritic cell (DC) vaccine achieves regulatory approval for clinical therapy. CD40-activated B cells have been tested in privately owned dogs presenting with NHL, and the results suggested that cell-based CD40 cancer vaccine is safe and synergizes with chemotherapeutic therapy to improve clinical outcome in canine NHL (Sorenmo et al., 2011). Generally, cancer antigens have weak immunogenicity and need effective vaccine adjuvant to prime specific immune responses. The new adjuvant includes CpG, ssRNA, monophosphoryl lipid A, saponins, heat shock family proteins, and cytokines, such as IFN-γ, interleukin (IL)-2, IL-12, and so on.

Ginseng is one of the most commonly used traditional Chinese medicines in the therapy of various human diseases. Ginseng contains much effective and active components to activate immune responses. For example, ginseng stem-leaf saponins had an adjuvant effect on an attenuated pseudorabies virus vaccine in mice model by increasing the expression of several miRNAs in macrophages, such as miR-132, miR-146a, miR-147, and miR-155 (Ni et al., 2016). The ginseng-fraction Rb1 induced serum-detectable amounts of IL-4 and IL-10 after the prime immunization, and 5 weeks after booster, the activated lymphocytes produced large amounts of cytokines, including IFN-γ, IL-2, IL-4, IL-10, and tumor necrosis factor-alpha (TNF-α), suggesting that Rb1 of ginseng could elicit mixed Th1 and Th2 immune responses (Rivera et al., 2005). Ginseng fruit polysaccharide obviously suppressed tumor growth and lung metastasis in vivo by promoting spleen lymphocyte proliferation, increasing natural killer (NK) cell activity, and upregulating the serum concentration of IL-2 and IFN-γ (Wang et al., 2015). Ginsenoside Rg1 is one of the active components of ginseng and it has various pharmacological effects, including antisenescent, neuroprotective, immune-stimulatory, and antidiabetic effects (Li et al., 2016; Anggelia and Chan, 2017; Tian et al., 2017). Rg1 exerts its regulatory mechanism in various diseases involving in several important signaling pathways, such as VEGF-C/VEGFR-3 signaling pathway (Yu et al., 2016), AMPK/mTOR/PI3K pathway (Mao et al., 2016), Akt-FoxO3a-Bim signaling pathway (Liu et al., 2016), NF-κB/NO pathway (Wu et al., 2016), and GR/BMP-2 signaling pathway (Gu et al., 2016). Ginsenoside Rg1 also regulated innate immune responses in macrophage by differentially regulating the NF-κB and PI3K/Akt/mTOR pathways (Wang et al., 2014). Moreover, ginsenoside Rg1 regulated adaptive immune responses as vaccine adjuvant to enhance the anti-infection effects against hepatitis B surface antigen in mice, and the adjuvant activities of Rg1 were involved in TLR4 signaling pathway (Yuan et al., 2016). The hemolytic activity of Rg1 was much lower compared with Quil-A, while ginsenoside Rg1 in combination with aluminum hydroxide (alum) synergistically promoted a mixed Th1/Th2 immune response to ovalbumin (OVA) in BALB/c mice (Sun et al., 2008). Ginsenoside Rg1 combined with the Toxoplasma gondii rSAG1 (recombinant surface antigen 1) triggered a stronger humoral and cellular response against T. gondii (Qu et al., 2011). Thus, Rg1 could be a promising vaccine adjuvant and worth further development. In the present study, we first detected the activities of Rg1 in human peripheral blood mononuclear cell (PBMC)-derived DCs and tested the vaccine adjuvant effects of Rg1 by injecting E.G7-OVA cells into C57BL/6 mouse. The study will give a new clue on the immune therapy of lymphoma patients.

Materials and Methods

Cell lines and agents

Recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) (Cat. No. 214-GM-010) and recombinant human IL-4 (Cat. No. 204-IL-010) were obtained from R&D Systems (Heidelberg, Germany). RPMI 1640 (Cat. No. 61870044) and fetal serum (Cat. No. 16000069) were purchased from Life Technologies (Invitrogen). E.G7-OVA cells were purchased from Reagent Technology Corp. (Shanghai, China; Cat. No. TW-CL-0316). Ginsenosides Rg1 was obtained from Hongjiu Biotechnology (Jilin, China). The antigen OVA protein (grade V; Sigma, St. Louis, MO) was purchased from Sigma Corporation. It was dissolved in 0.15 M NaCl and filter sterilized before injection.

Generation of human DCs

Peripheral blood mononuclear cells were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation by Ficoll-Hypaque (Sigma) density gradient centrifugation. Human peripheral blood monocyte-derived DCs were generated as described with a few modifications (Huck et al., 2005; Li et al., 2011b). Briefly, PBMCs were cultured in six-well plates at 3 × 10⁶ cells/well in 4 mL of complete medium (RPMI 1640 supplemented with 100 U/mL penicillin, 100 μg/mL of streptomycin, and 10% fetal calf serum [all from Life Technologies, Invitrogen]) in the presence of recombinant human GM-CSF (50 ng/mL) and recombinant human IL-4 (30 ng/mL). Medium was changed every other day and supplemented with new cytokines. The cells were cultured for 5 days to form immature DCs.

DC cytokine and chemokine production

Human PBMC-derived DCs were adjusted to 4 × 10⁵ cells/mL for stimulation by Rg1; then, 0.1, 1.0, and 10 μg/mL of Rg1, 20 ng/mL lipopolysaccharides (LPS) (Sigma) or phosphate-buffered saline (PBS) were added. Supernatants were harvested after 48 h, and the concentration of DC cytokines, such as IL-6, IL-1β, and TNF-α, and chemokines, such as IFN-γ-inducible protein 10 and IL-8, were determined with enzyme-linked immunosorbent assay (ELISA) kits (Neobioscience, Beijing, China) according to kit protocols.

Flow cytometry analysis

Cultured for 5 days, human DCs were stimulated with 10 μg/mL of Rg1 or PBS for 48 h and then collected and washed with PBS. For fluorescence-activated cell sorting (FACS) analysis, the cells were stained with phycoerythrin-conjugated anti-CD80, anti-CD83, and or FITC-conjugated anti-CD14, anti-HLA-DR monoclonal antibody (mAb; BioLegend) for analysis by a FACSCalibur Flow Cytometer (Becton Dickinson, Mountain View, CA) and CellQuest software (Becton Dickinson).

Mice and immunization

Six- to 8-week-old male C57BL/6 mice were kept in specific pathogen-free conditions. They were weighed and randomly divided into four groups each as follows: Rg1-OVA group, OVA group, Rg1 group, and PBS group. They were intraperitoneally immunized twice, 2 weeks apart, with 10 μg OVA combined with 10 μg of Rg1, 10 μg OVA, 10 μg Rg1 or PBS in a volume of 100 μL/mouse. Each group contained 10 mice. The experiment was approved by the Institutional Animal Care and Use Committee of Zhangzhou Affiliated Hospital of Fujian Medical University.

Splenocyte proliferation and cytokine release

Mouse splenocytes were prepared as described (Li et al., 2011a). Splenocytes (5 × 10⁵/mL) were cultured at 37°C in the presence of 5% CO₂, with or without 10 μg/mL OVA_257–264, OVA, BSA, or ConA. After 48 h incubation, splenocyte proliferation in each group was tested by MTT assay. For cytokine detection, culture supernatant was harvested, and the presence of IFN-γ and IL-2 was tested with commercial mouse ELISA kits (Neobioscience). The concentrations of IFN-γ in the samples were calculated from standard curves.

Tumor challenge

C57BL/6 mice were immunized as described above. Seven days after the final immunization, C57BL/6 mice were challenged subcutaneously with 3 × 10⁵ E.G7-OVA tumor cells in the flank area. Body weight was measured every 3 days, and survival following tumor challenge was recorded. Survival rate was calculated by GraphPad 5.0.

Statistical analyses

The survival rate in each group was analyzed by log-rank (Mantel–Cox) test. The other statistical analyses were based on Student's t-test. The results are shown as mean value ± standard deviation. p < 0.05 was considered as statistically different.

Results

Rg1 promotes phenotypic maturation of human PBMC-derived DCs

To determine whether Rg1 induced the maturation of human immature dendritic cells (iDCs), FACS assay was performed to determine the expression of surface makers on the PBMC-derived DCs. Human PBMC-derived iDCs were prepared as described in Materials and Methods section, and the iDCs were treated with 10 μg/mL of Rg1 for 48 h. The expression of a maturation marker CD83, costimulatory molecules CD80, and human leukocyte antigen – antigen D related (HLA-DR), as well as the surface marker of macrophage CD14, is shown in Figure 1. The data revealed that the expression of HLA-DR and CD83 was significantly higher in Rg1-treated DCs than that in iDCs (**p < 0.01). The data showed that Rg1 induced a moderate expression of CD80 in human DCs and low expression of CD14, suggesting that Rg1 promoted the phenotypic maturation of the human iDCs (Fig. 2).

FIG. 1.

The expression of DCs' cell surface marker is determined by fluorescence-activated cell sorting analysis. Human peripheral blood monocytes were induced into immature DCs as described in Materials and Methods section. (A) The untreated DCs and isotype controls, including IgG2α-FITC and IgG1, κ-PE, were shown. (B) The iDCs were treated with 10 μg/mL of Rg1 for 48 h. (C) The immature DCs were used as negative controls. The CD14, CD80, HLA-DR, and CD83 expression were measured by flow cytometry. Results are representative of at least three independent experiments. DC, dendritic cell; HLA-DR, human leukocyte antigen – antigen D related; iDCs, immature dendritic cells.

FIG. 2.

Rg1 promotes phenotypic maturation of human PBMC-derived DCs. (A) Mean fluorescence intensities from three independent experiments. The immature DCs were used as negative controls. MFI levels were higher in Rg1-treated DCs than that in immature DCs (**p < 0.01). (B) Cell morphology under an optical microscope at first day and seventh day (magnification 100 × ). PBMCs were cultured as described in Materials and Methods section. Briefly, the cells were cultured in RPMI 1640 medium supplemented with10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL of streptomycin, 50 ng/mL of recombinant human GM-CSF, and 30 ng/mL of recombinant human IL-4. Medium was changed every other day and supplemented with new cytokines. The cells were cultured for 5 days to form immature DCs. GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; PBMC, peripheral blood mononuclear cell.

Rg1 promotes cytokine secretion by human PBMC-derived iDCs

To test the effects of Rg1 to stimulate the secretion of cytokines, immature DCs were treated with increasing concentrations of Rg1 for 48 h, and the levels of cytokines were determined in the harvested supernatants by ELISA assay. As shown in Figure 3, the immature DCs were treated with 0.1, 1.0, and 10 μg/mL of Rg1 for 48 h, and the results demonstrated that Rg1 significantly increased the secretion of TNF-α, IL-1β, and IL-6 in a dose-dependent manner. All the results revealed that Rg1 induced cytokine secretion of human PBMC-derived DCs.

FIG. 3.

Rg1 promotes cytokine secretion by human PBMC-derived iDCs. The immature DCs were prepared as described in Materials and Methods section. The iDCs were treated with Rg1 at the concentration of 0.1, 1.0, and 10 μg/mL for 48 h. The levels of TNF-α, IL-1β, and IL-6 were determined by ELISA assay. Data are shown as mean value ± standard deviation. **p < 0.01 for the difference between TNF-α, IL-1β, and IL-6 production in the Rg1-treated and untreated cells. ELISA, enzyme-linked immunosorbent assay; TNF-α, tumor necrosis factor-alpha.

Rg1 induces chemokine secretion by human PBMC-derived DCs

To determine the effects of Rg1-treated DCs to recruit effector cells to inflammatory sites, ELISA assay was performed to determine the levels of chemokines in Rg1-treated cells, LPS-treated cells, and untreated cells. LPS-treated cells were used as positive controls, and untreated cells were used as negative controls. As shown in Figure 4, the immature DCs were treated with 10 μg/mL of Rg1 for 24 h, and the results showed that the secretion of IL-8 and IFN-γ-inducible protein 10/IP-10/CXCL10 was significantly higher by Rg1-treated cells than that by untreated DCs. Moreover, we used increasing concentrations of Rg1 to treat iDCs for 24 h and 48 h, respectively. The data showed that the levels of IL-8 and IP-10 were significantly increased in a dose-dependent manner (**p < 0.01, compared with untreated cells for 48 h, ^## p < 0.01, compared with untreated cells for 24 h).

FIG. 4.

Rg1 induces chemokine secretion by human PBMC-derived DCs. (A) The immature DCs were treated with 10 μg/mL of Rg1 for 24 h, the concentration of chemokines IL-8 and IP-10 was determined by ELISA assay. LPS was used as positive control. **p < 0.01, compared with untreated cells. (B) The immature DCs were treated with increasing concentrations of Rg1 for 24 and 48 h. The concentration of IL-8 and IP-10 was determined by ELISA assay. **p < 0.01, compared with untreated cells in 48 h; ^## p < 0.01, compared with untreated cells in 24 h. LPS, lipopolysaccharides.

Rg1 induced antigen specific splenocyte proliferation C57BL/6 mice were randomly divided into four groups and immunized twice with 10 μg of Rg1, 10 μg of OVA, 10 μg of Rg1 plus 10 μg of OVA, or PBS, respectively. Ten days after the final immunization, splenocytes were prepared and stimulated with 1 μg/mL of OVA_257–264, OVA, unrelated control protein BSA, and ConA for 24 h. The cell viability of splenocytes in each immunized group was determined by MTT assay. As shown in Figure 5A, after in vitro stimulation with OVA_257–264 and OVA protein for 24 h, the splenocytes from the mice immunized with OVA in combination with Rg1 were more proliferative than in either control group (p < 0.01). In addition, no differences were observed between splenocytes from OVA mice and PBS mice when the cells were treated with OVA. The possible explanation was that OVA immunogenicity was too weak to prime effectively OVA-specific splenocyte proliferation.

FIG. 5.

Rg1 induces antigen specific splenocyte proliferation and CTL activity. The mice were immunized as described in Materials and Methods section. Seven days after final immunization, splenocytes were isolated and stimulated in vitro with OVA_257–264, OVA, unrelated control protein BSA, or positive control ConA for 24 h. The splenocyte proliferation activity was determined by MTT assay. **p < 0.01, compared with OVA-immunized mice. CTL, cytotoxic lymphocyte; OVA, ovalbumin.

The secretion of IFN-γ and IL-2 is increased in mice immunized with Rg1 plus OVA

To identify whether Rg1 could induce Th1-type immune response such as secreting Th1 cytokines in animal model, ELISA assay was performed to test the concentration of IFN-γ and IL-2 in the supernatant of splenocytes in each group. As shown in Figure 6, the splenocytes in each group were in vitro stimulated with 1 μg/μL of OVA_257–264 peptide for 24 h, and the data revealed that the mice immunized with Rg1 and OVA produced higher level of IFN-γ and IL-2 compared with OVA immunized group (**p < 0.01).

FIG. 6.

The mice immunized with Rg1 and OVA produce Th1-type cytokines. The mice were immunized as described in Materials and Methods section. After final immunization, the splenocytes in each group were stimulated with 1 μg/μL of OVA_257–264 peptide for 24 h. The concentration of IFN-γ (A) and IL-2 (B) in supernatant was determined by ELISA assay. **p < 0.01, compared with OVA immunized mice.

Rg1-OVA immunized mice inhibits E.G7-OVA tumor cell growth

E.G7-OVA was a OVA-expressing tumor cell line with parental EL4 cells. To detect the in vivo antitumor immunity by Rg1-OVA immunization in murine model, E.G7-OVA cells were injected in each group immunized with Rg1-OVA, Rg1, OVA, or PBS, respectively. The tumors were photographed at 21 days after final immunization (Fig. 7A). Importantly, mice immunized with Rg1 plus OVA showed significant tumor growth inhibition (p-value = 0.0117, generalized log-rank [Mantel–Cox] test). There was no significant difference in survival among the other control groups (Fig. 7B). No tumors were observed until 2 weeks after E.G7-OVA tumor challenge in OVA immunized group, Rg1 immunized group, and PBS group, but no tumors were found in Rg1-OVA group until 27 days after tumor challenge. All of the data suggested that immunization of mice with Rg1-OVA induced a potent and protective immune response against OVA-expressing tumor cells.

FIG. 7.

The mice immunized with Rg1 and OVA inhibits tumor growth of E.G7-OVA.C57BL/6 mice were immunized subcutaneously with Rg1-OVA, OVA, Rg1, and PBS, respectively. The mice were challenged subcutaneously after final immunization with 1 × 10⁶ E.G7-OVA tumor cells to the flank area of the thigh. (A) The representative of immunized mice in Rg1 group, OVA group, OVA-Rg1 group, and PBS group. The tumors were photographed at 21 days after final immunization. (B) Survival rate of immunized mice after E.G7-OVA challenge was recorded. Each group contained six mice. PBS, phosphate-buffered saline.

Discussion

DCs are the major professional antigen-presenting cells. The effective vaccine is first to activate DCs and prime the adaptive immune responses. Cancer antigens are usually with weak immunogenicity and unable to prime effective antitumor responses. Immunoadjuvant is capable to induce DC maturation and enhance cellular immunity in immunotherapy for human cancers. Phase II trial of adjuvant immunotherapy with autologous tumor-derived Gp96 vaccination demonstrated to be a safe way in therapy, and tumor-specific immune responses were elicited in patients with gastric cancers (Zhang et al., 2017). Mycobacterium tuberculosis Rv0652-derived adjuvant obviously promoted DC-based tumor immunotherapy (Lee et al., 2014). Listeria-derived ActA was also reported to be an effective adjuvant for primary and metastatic tumor immunotherapy (Wood et al., 2010). Thus, immunotherapy was a promising method in the therapy of human cancers. In the present study, we first tested the adjuvant effects of Rg1 on PBMC-derived DCs and further explored the antitumor activity of Rg1 combined with OVA in the mouse model. We found that Rg1, as cancer vaccine adjuvant, was effective to activate innate and adaptive immune responses. Especially, Rg1 could activate the PBMC-derived DCs and promote adaptive immune responses to exert anticancer effects in C57BL/6 mice.

First, Rg1 had the ability to activate the PBMC-derived DCs by promoting the production and secretion of cytokines (such as TNF-α, IL-1β, and IL-6) and chemokines (such as IL-8, IP-10). The secretion of pro-inflammatory cytokines could activate human granulocytes and recruit a variety of the immune cells, including monocytes and natural killer cells into inflammatory site and prime innate inflammatory responses. Moreover, the chemokine production such IL-8 could enhance the migration of DCs and macrophages. IP-10 was critical for effector T cell generation and induced Th1-type immune responses. PBMC-derived DCs were treated with increasing concentrations of Rg1, and we found that Rg1 increased the cytokines and chemokine production in a dose-dependent manner. All the results demonstrated that Rg1 activated innate immune response by activating the function of DCs. Rg1 obviously promoted the secretion of cytokines and chemokines, such as TNF-α, IL-1β, IL-6, and IL-8. We speculated that the molecular mechanism of Rg1 in promoting the activation of DCs was probably by activating the NF-κB signaling pathway (Wu et al., 2016). However, the exact molecular mechanism was to be further clarified. Moreover, Rg1 induced the phenotypic maturation of human PBMC-derived iDCs. As we have known, MHC molecule was responsible to present antigen to effector T cells, and the costimulatory molecule was the secondary signal to activate T cells. In this study, FACS assay results revealed that the maturation marker CD83, costimulatory molecule CD80, and HLA-DR had a higher expression in Rg1-treated DCs than that in untreated iDCs, suggesting that Rg1 had a promising potential to prime adaptive immune response by activating the antigen-processing cells.

Besides inducing DCs to produce cytokines and chemokines, Rg1 played a crucial role as vaccine adjuvant on protein immunization. EL4 cells were derived from T lymphocytes from C57BL/6 (H-2 b) mouse with a high degree of malignancy. E.G7-OVA cells were derived from EL4 lymphoma cells transfected by electroporation with the plasmid pAc-neo-OVA carrying a complete copy of chicken OVA mRNA and the neomycin (G418) resistance gene, which led to constitutive synthesis and secretion of OVA from E.G7-OVA cells. In the study, we used OVA as antigen and Rg1 as adjuvant in C57BL/6 mice. In this study, Rg1 induced antigen-specific splenocyte proliferation and upregulated the production of IFN-γ and IL-2 in the splenocytes of the mice immunized with Rg1 and OVA. IFN-γ and IL-2 were the major potent cytokines, and the results demonstrated that Rg1 induced Th1-response in Rg1-OVA immunized mice. We also tested the cytotoxic lymphocyte (CTL) responses after the final immunization in the animal model and the results demonstrated that OVA-specific CTL activity was observed when in vitro stimulated with OVA_257–264 or OVA protein in the Rg1 and OVA-immunized mice (data were not shown). Importantly, the OVA-specific antitumor activity was observed in E.G7-OVA challenge model, and the results demonstrated that the survival rate of mice immunized with OVA combined with Rg1 was significantly higher compared with the other group of mice. All the results demonstrated that Rg1 combined with OVA primed an important antitumor immunity in murine model. Thus, Rg1 possessed a potent adjuvant effect on cancer vaccine and elicited a specific Th1-response in antitumor immunity, which could have potential as a prophylactic vaccine adjuvant to control lymphomas.

Footnotes

Acknowledgment

The work was supported by Fujian provincial health system in the youth backbone personnel training program (Grant No. 2014-ZQN-JC-36).

Disclosure Statement

No competing financial interests exist.

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