Polysaccharide Isolated From Tetrastigma hemsleyanum Activates TLR4 in Macrophage Cell Lines and Enhances Immune Responses in OVA-Immunized and LLC-Bearing Mouse Models

Abstract

Tetrastigma hemsleyanum Diels et Gilg is a valuable Chinese medicinal herb with a long history of clinical application. Our previous study isolated and characterized a purified polysaccharide from the aerial part of Tetrastigma hemsleyanum (SYQP) and found it having antipyretic and antitumor effects in mice. A preliminary mechanistic study suggests these effects may be related to the binding of toll-like receptor (TLR4). The objective of this study is to further explore the detailed stimulating characteristics of SYQP on TLR4 signaling pathway and its in vivo immune regulating effect. We use HEK-BLUE hTLR4, mouse and human macrophage cell lines, as research tools. In vitro results show SYQP activated HEK-BLUE hTLR4 instead of HEK-BLUE Null cells. The secretion and the mRNA expression of cytokines related to TLR4 signaling significantly increased after SYQP treatment in both PMA-induced THP-1 and RAW264.7 macrophage cell lines. The TLR4 antagonist TAK-242 can almost completely abolish this activation. Furthermore, molecules such as IRAK1, NF-κB, MAPKs, and IRF3 in both the MyD88 and TRIF branches were all activated without pathway selection. In vivo results show SYQP enhanced antigen-specific spleen lymphocyte proliferation and serum IgG levels in OVA-immunized C57BL/6 mice. Orally administered 200 mg/kg SYQP induced obvious tumor regression, spleen weight increase, and the upregulation of the mRNA expression of TLR4-related cytokines in Lewis lung carcinoma-bearing mice. These results indicate SYQP can act as both a human and mouse TLR4 agonist and enhance immune responses in mice (p < 0.05). This study provides a basis for the development and utilization of SYQP as a new type of TLR4 agonist in the future.

Keywords: RAW264.7; THP-1; TLR4; Tetrastigma hemsleyanum; adjuvant; antitumor; polysaccharide.

FIGURE 1

SYQP stimulated human TLR4 in HEK-BLUE hTLR4 cells. (A) SYQP could activate NF-κB in HEK-BLUE hTLR4 but not HEK-BLUE NULL cells. (B) SYQP showed no cytotoxicity. Cells were treated with SYQP for 24 h, and cell viability was evaluated by MTS assay. (C) SYQP pretreatment could not inhibit LPS-induced NF-κB activation in HEK-BLUE hTLR4 cells. Cell culture supernatants were mixed with QUANTI-BLUE substrate to test the level of SEAP, which represented the activity of NF-κB in the HEK-BLUE reporter cells. All of the data were normalized to the 1 μg/ml LPS-treated group, which represented the maximum response of the cells. All of the results are presented as the mean ± SD (n = 3).

FIGURE 2

SYQP stimulated human TLR4 in PMA-induced THP-1 cells. (A) Effects of SYQP on viability of THP-1 cells. Cells were treated with SYQP for 24 h, and cell viability was evaluated by MTS assay. (B) SYQP increased the secretion of TNF-α from THP-1 cells. The cells were treated with SYQP at the indicated concentration for 24 h. Cell culture medium was collected, and the secretion levels of cytokines were detected by ELISA. ***p < 0.001 vs. control (the concentration of SYQP was zero). (C) TLR4 antagonist TAK-242 decreased the secretion of TNF-α promoted by SYQP. The cells were treated with 100 μg/ml SYQP alone or combined with TAK-242 for 24 h. LPS at 1 μg/ml was used as a positive control. Cell culture medium was collected, and the secretion levels of TNF-α were detected by ELISA. ***p < 0.001 vs. cells treated without TAK-242. All of the results are presented as the mean ± SD (n = 3). (D) SYQP induced mRNA expression of both MyD88-dependent and TRIF-dependent cytokines in THP-1 cells. The cells were treated with 1, 10, 100 μg/ml SYQP or 1 μg/ml LPS for 6 h. After that, mRNA was isolated, and the gene expression levels of cytokines were determined by real-time PCR. The results are presented as the mean ± SD (n = 3). **p < 0.01, ***p < 0.001 vs. control.

FIGURE 3

SYQP stimulated mouse TLR4 in RAW264.7 cells. (A) Effects of SYQP on the viability of RAW264.7 cells. (B) SYQP increased the secretion of TNF-α from RAW264.7 cells. The cells were treated with SYQP at the indicated concentration for 24 h. Cell culture medium was collected, and the secretion levels of cytokines were detected by ELISA. ***p < 0.001 vs. control. (C) TAK-242 decreased the secretion of TNF-α promoted by SYQP. ***p < 0.001 vs. cells treated with and cells treated without TAK-242. (D) SYQP upregulated mRNA expression of both MyD88- and TRIF-dependent cytokines in RAW264.7 cells. The cells were treated with 1, 10, 100, 500 μg/ml SYQP or 1 μg/ml LPS for 6 h. After that, mRNA was isolated, and the gene expression levels of cytokines were determined by real-time PCR. **p < 0.01, ***p < 0.001 vs. control. All of the results are presented as the mean ± SD (n = 3).

FIGURE 4

SYQP activated pathways downstream of TLR4 without selection. (A) RAW264.7 cells were treated with 1–500 μg/ml SYQP or 1 μg/ml LPS for 2 h (TLR4, IRAK1, IRF3) or 30 min (IKK, NF-κB, p38, JNK and ERK). (B) RAW264.7 cells were treated with 100 μg/ml SYQP or 1 μg/ml LPS for the indicated time points. The expression of molecules in the TLR4 pathways was determined by western blot analysis. The blot shown is a representative of one of three similar experiments. The densitometric analysis of the protein expression was shown in Supplementary Figure S2.

FIGURE 5

TAK-242 inhibited the phosphorylation of pIRF3 and pNF-κBp65 induced by SYQP. The RAW264.7 cells were pretreated with TAK-242 20 μg/ml for 30 min; then, SYQP 100 μg/ml or LPS 1 μg/ml were added into the cell supernatant alone or combined with TAK-242 for another 2 h. The cell lysates were analyzed by western blot. (A) The protein expression of TLR4, pIRF3, IRF3, NF-κBp65, and pNF-κBp65. (B) Densitometric analysis of the protein expression. The data are presented as the mean ± SD from three independent experiments.

FIGURE 6

SYQP enhanced antigen-specific immune responses in OVA-immunized mice models. C57BL/6 mice were subcutaneously immunized with OVA (100 µg) at weeks 0 and 3. SYQP was given at 50, 100, and 200 mg/kg by intragastric administration once a day for four consecutive days before each immunization. Two weeks after the second immunization, blood samples were collected for measurement of OVA-specific IgG by ELISA. Splenocytes were harvested and exposed to OVA 100 μg/ml again for 48 h to determine antigen-specific lymphocyte proliferation by MTS assay. Stimulation index = (OD _{OVA exposure} − Blank)/(OD _{OVA non-exposure} − Blank). (A) SYQP enhanced specific serum IgG levels. (B) SYQP enhanced the spleen lymphocyte stimulation index. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (OVA-immunized mice without SYQP treatment).

FIGURE 7

SYQP reduced tumor burden and enhanced spleen weight of LLC-bearing mice. C57BL/6 mice (n = 6) were injected s.c. with LLC tumor cell lines. Once palpable tumors were formed (day 7), mice were orally administered with SYQP (100, 200 mg/kg) or PBS (control) for seven consecutive days. DDP at 2 mg/kg was intraperitoneally injected once every other day and set as a positive control. On day 15, mice were killed, and tumor tissues and spleen were excised and weighed. (A) SYQP administration reduced the tumor burden of LLC-bearing mice. (B) SYQP administration increased the spleen weight of LLC-bearing mice. (C) The effect of SYQP on the mRNA expression of TLR4, TNF-α, and IP-10 in the spleen tissue. *p < 0.05, ***p < 0.001 vs. control.