Rehmannia glutinosa polysaccharide induces toll-like receptor 4 dependent spleen dendritic cell maturation and anti-cancer immunity

Abstract

Rehmannia glutinosa polysaccharide (RGP) has shown an activation of immune cells in vitro. However, the immune stimulatory effect of RGP in a mouse in vivo is not well studied. In this study, we examined the effect of RGP on dendritic cell (DC) activation and anticancer immunity in vivo. Treatments of RGP in C56BL/6 mice induced increased levels of co-stimulatory molecule expression and pro-inflammatory cytokine production in spleen DCs dependent on toll-like receptor 4 (TLR4), and those DCs promoted interferon-gamma (IFNγ) production in CD4⁺ and CD8⁺ T cells. RGP also enhanced ovalbumin (OVA) antigen (Ag)-specific immune activation in tumor-bearing mice, including Ag presentation in DCs, OT-I and OT-II T-cell proliferation, migration of OT-I and OT-II T cells into the B16-OVA tumor, OVA-specific IFNγ production, and the specific killing of OVA-coated splenocytes, which consequently inhibited B16-OVA tumor growth dependent on TLR4 and CD8⁺ T cells. Finally, the combination of RGP and self-Ag treatment efficiently inhibited CT26 carcinoma and B16 melanoma tumor growth in BLAB/c and C57BL/6 mice, respectively. These data demonstrate that RGP could be a useful adjuvant molecule for immunotherapy against cancer.

Keywords: Adjuvant; Rehmannia glutinosa polysaccharide; TLR4; anticancer; dendritic cell maturation.

Figure 1.

RGP-induced activation of BMDCs and spleen DCs. BMDCs were treated with 10, 50, and 100 μg/mL RGP and with 2 μg/mL LPS for 24 h. (A) Morphological changes of BMDCs. (B) Mean fluorescence intensity levels of CD40, CD80, CD86, MHC class I, and MHC class II in the BMDCs. (C) C57BL/6 mice were injected intravenously (i.v.) with 12.5, 25, and 50 mg/kg RGP and with 1 mg/kg LPS. The flow cytometric analyses of co-stimulatory molecules and MHC class I and II in gated lineage-CD11c+ cells from the spleen are shown. (D) Concentrations of IL-6, IL-12p40, and TNF-α in BMDC-cultured medium. (E) The serum concentration of IL-6, IL-12p40, and TNF-α in RGP-treated mouse is shown. (F) C57BL/6 mice were injected i.v. with 50 mg/kg RGP. Three days later, the mice were injected again with the same amount of RGP, and then 3 d later, intracellular IFNγ, IL-4, and IL-17 in CD4⁺ and CD8⁺ T cells in the spleen were analyzed with flow cytometry. All data are representative of the average of analyses of six independent samples (two mice per experiment, total three independent experiments). *p < 0.05, **p < 0.01.

Figure 2.

RGP-induced activation spleen DCs were dependent on TLR4. C56BL/6 wild-type, TLR2-KO, TLR4-KO, and SR-A-KO mice were treated i.v. with PBS and 50 mg/kg RGP for 24 h. (A) Co-stimulatory molecules and MHC class I and II were measured in the spleen DCs. (B) The serum concentrations of IL-6, IL-12p40, and TNF-α after RGP treatment are shown. (C) The mRNA levels of IFNγ and T-bet in the spleen are shown. All data are the average of analyses of six independent samples (two mice per experiment, total three independent experiments). **p < 0.01.

Figure 3.

RGP-enhanced OVA presentation in DCs and OVA-specific T cell proliferation. C57BL/6 mice were injected i.v. with PBS, 2.5 mg/kg OVA, 50 mg/kg RGP, and the combination of RGP and OVA. The combination of LPS and OVA also injected to mice as a positive control. (A) The surface presentation of OVA Ag on the spleen CD8α⁺ and CD8α⁻ DCs was measured by anti-OVA (257–264) antibodies 24 h after RGP treatment (left panel). Mean positive cells of OVA peptide on the surface of the spleen CD8α⁺ and CD8α⁻ DCs are shown (right panel). (B) The proliferation of adaptive transferred CSFE-labeled OT-I and OT-II cells in the CD45.1 congenic mice was analyzed with flow cytometry (left panel). Mean proliferating cells in OT-I and OT-II are shown (right panel). All data are representative of the average of analyses of six independent samples (three mice per experiment, total two independent experiments). *p < 0.05, **p < 0.01.

Figure 4.

RGP-promoted DC maturation in the tumor microenvironment. C57BL/6 mice were injected subcutaneously (s.c.) with 1 × 10⁶ B16 melanoma cells or B16-OVA cells. Fifteen days after tumor injection, the mice were treated with PBS, 50 mg/kg RGP and 1 mg/kg LPS for 24 h. (A) MFI of CD40, CD80, CD86, and MHC class I and II levels was measured in the spleen and tumor drLN DCs. (B) Concentrations of IL-6, IL-12p40, and TNF-α in serum. (C) Surface OVA peptide (257–264) presentation was measured in the tumor drLN DCs after treatment of PBS, 50 mg/mL RGP, 1 mg/kg LPS with or without 2.5 mg/kg OVA (left panel). Mean positive cells of OVA peptide presenting DCs are shown (right panel). (D) CFSE-labeled OT-I and OT-II T-cell proliferation in B16 tumor-bearing CD45.1 congenic mice were analyzed with flow cytometry. (E) Percentage of IFNγ⁺ and TNF-α⁺ cells in B16-OVA tumor-infiltrated OT-I and OT-II cells. (F) OT-I and OT-II T-cell proliferation in wild-type and TLR4-KO mice. (G) Intracellular IFNγ- and TNF-α-producing OT-I and OT-II cells in B16-OVA tumor in the wild-type and TLR4-KO mice. All data are representative of the average of analyses of six independent samples (three mice per experiment, total two independent experiments). **p < 0.01, *p < 0.05.

Figure 5.

The combination of RGP and OVA treatment prevented B16-OVA tumor growth. C57BL/6 mice were injected s.c. on the right side with 1 × 10⁶ B16-OVA cells. Once tumors were well established on day 7, the mice received s.c. injections of PBS, 2.5 mg/kg OVA, 50 mg/kg RGP, the combination of OVA and RGP and the combination of OVA and LPS as a positive control. On day 14, the mice received the same treatment again. (A) The curves of B16-OVA tumor growth in mice are shown. (B) The size of the tumor masses on day 21 after B16-OVA tumor cell challenge. (C) OVA peptide (257–264)- and (323–339)-specific IFNγ production was analyzed by ELISPOT analysis (Upper panel). The mean number of spots is shown (lower panel). (D) Cytotoxic T lymphocyte (CTL) activity was assessed in vivo on day 21 by adoptive transfer of CFSE-labeled and SIINFEK-loaded splenocytes, and also of a control splenocyte population without peptide labeled with CMTMR. Dot plots show the percentage of SIINFEK-loaded CFSE⁺ cells and non-peptide-loaded CMTMR⁺ cells (upper panel). Mean percentages of Ag-specific lysis (lower panel). (E, F) Antitumor effect of the combination of OVA and RGP were measured in (E) CD8⁺ T cell-depleted and (F) TLR4-KO mice. Data are from analyses of six individual mice (three mice per experiment, total two independent experiments). **p < 0.01.

Figure 6.

RGP promoted self-antigen (Ag)-specific immune activation and antitumor immunity. BLAB/c mice were injected s.c. with 1 × 10⁶ CT26 carcinoma cells. The mice were treated with PBS, 2.5 mg/kg AH1A5, 50 mg/kg RGP, and the combination of AH1A5 and RGP on days 7 and 14 of tumor injection. C57BL/6 mice were inoculated s.c. with 1 × 10⁶ B16 melanoma cells. Once tumors were well established, the mice received PBS, 2.5 mg/kg TRP2, 50 mg/kg RGP, and the combination of RGP and TRP2 on days 7 and 14 of tumor injection. The growth curves of (A) CT26 tumor in BALB/c and (B) B16 tumor in C57BL/c are shown. Data are the average of analyses of six independent samples (two mice per experiment, total three independent experiments). **p < 0.01. (C) Tumor masses of CT26 carcinoma and (D) B16 melanoma are shown. (E) AH1A5-specific IFNγ production in splenocyte of BALB/c mice and (F) TRP2-specific IFNγ production in the splenocytes of C57BL/c mice were analyzed by ELISPOT analysis (upper panel). The mean number of spots is shown (lower panel). All data are representative of the average of analyses of six independent samples (three mice per experiment, total two independent experiments). **p < 0.01.