Adjuvant activity of tubeimosides by mediating the local immune microenvironment

Abstract

Rhizoma Bolbostemmatis, the dry tuber of Bolbostemma paniculatum, has being used for the treatment of acute mastitis and tumors in traditional Chinese medicine. In this study, tubeimoside (TBM) I, II, and III from this drug were investigated for the adjuvant activities, structure-activity relationships (SAR), and mechanisms of action. Three TBMs significantly boosted the antigen-specific humoral and cellular immune responses and elicited both Th1/Th2 and Tc1/Tc2 responses towards ovalbumin (OVA) in mice. TBM I also remarkably facilitated mRNA and protein expression of various chemokines and cytokines in the local muscle tissues. Flow cytometry revealed that TBM I promoted the recruitment and antigen uptake of immune cells in the injected muscles, and augmented the migration and antigen transport of immune cells to the draining lymph nodes. Gene expression microarray analysis manifested that TBM I modulated immune, chemotaxis, and inflammation-related genes. The integrated analysis of network pharmacology, transcriptomics, and molecular docking predicted that TBM I exerted adjuvant activity by interaction with SYK and LYN. Further investigation verified that SYK-STAT3 signaling axis was involved in the TBM I-induced inflammatory response in the C2C12 cells. Our results for the first time demonstrated that TBMs might be promising vaccine adjuvant candidates and exert the adjuvant activity through mediating the local immune microenvironment. SAR information contributes to developing the semisynthetic saponin derivatives with adjuvant activities.

Keywords: adjuvant; molecular docking; network pharmacology; structure-activity relationships; transcriptomics; tubeimosides.

Figure 1

Chemical structure of three tubeimosides isolated from Rhizoma Bolbostemmatis. The chemical structures were identified by HR-ESI-MS and NMR. Ara, α-L-arabinopyranosyl; Glc, β-D-glucopyranosyl; Rha, α-L-rhamnopyranose; Xyl, β-D-xylopyranosyl.

Figure 2

Tubeimosides potentiated humoral and cellular immune responses to OVA in mice. The serum and spleen were collected 2 weeks after the booster immunization. (A) Serum OVA-specific antibody titers by ELISA. (B) Splenocyte proliferation by the MTT method (C) NK cell activities by the MTT method. (D) DTH in the OVA-sensitized ICR mice. The data were presented as mean ± SD (n = 5). ^a P < 0.05, ^b P < 0.01, and ^c P < 0.001 vs OVA.

Figure 3

TBM I elicited both Th1/Th2 and Tc1/Tc2 responses towards OVA in mice. Splenocyte and culture supernatants were collected 72 h after stimulation with OVA. (A) The contents of cytokines in culture supernatants using ELISA kits. (B) The mRNA expression levels of cytokines and TFs in OVA-stimulated splenocytes by RT-qPCR. (C–E) Splenocytes were subjected to flow cytometry for measuring OVA-specific T cell cytokine response. (C) Schematic diagram of gating on IFN-γ⁺ and IL-4⁺ CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. Frequencies of OVA-specific CD4 (D) and CD8 (E) T cells expressing single cytokine. The data are presented as mean ± SD (n = 5). ^b P < 0.01, and ^c P < 0.001 vs OVA.

Figure 4

TBM I elicited cytokine and chemokine expression at the injection site. Mice were injected i.m. with TBM I and mock contralateral muscle received PBS. (A) The levels of cytokines and chemokines in quadriceps muscles at 3, 6, and 12 hpi by ELISA. (B) The gene expression levels of cytokines and chemokines in quadriceps muscles at 2, 4, and 6 hpi using RT-qPCR. The data are presented as mean ± SD (n = 5). ^a P < 0.05, ^b P < 0.01, and ^c P < 0.001 vs OVA.

Figure 5

TBM I promoted the recruitment and antigen uptake of immune cells at the injection site. Mice were injected i.m. with OVA-AF488 alone or adjuvanted with TBM (I) The quadricep muscle tissues were collected at the indicated time points and subjected to FCM. (A) The number of immune cells recruited into the quadricep muscles at 12, 24, and 48 hpi. (B) The number of Ag⁺ immune cells recruited into the quadricep muscles at 12, 24, and 48 hpi. The data are presented as means ± SD (n = 4). ^a P < 0.05, ^b P < 0.01, and ^c P < 0.001 vs OVA-AF488. DC, dendritic cell; NEUT, neutrophil; MONO, monocyte; MΦ, macrophage; EOS, eosinophil; MC, mast cell; BASO, basophil.

Figure 6

TBM I induced the migration of immune cells and antigen transport to dLNs. Mice were injected i.m. with OVA-AF488 alone or adjuvanted with TBM (I) The inguinal lymph nodes were collected at the indicated time point and subjected to FCM. (A) The number of immune cells migrated to dLNs at 12, 24, and 48 hpi. (B) The antigen ingestion of DCs, monocytes, macrophages, and B cells in dLNs at 12, 24, and 48 hpi by cell numbers × MFI. The data are presented as mean ± SD (n = 4). ^a P < 0.05, ^b P < 0.01, and ^c P < 0.001 vs OVA-AF488. DC, dendritic cell; NEUT, neutrophil; MONO, monocyte; MΦ, macrophage; EOS, eosinophil; MC, mast cell; BASO, basophil.

Figure 7

Gene set enrichment analysis of gene expression profiles in mouse quadricep muscles induced by TBM I and hub genes screening. (A) Volcano plots of gene expression in quadricep muscles. (B) GO function and KEGG pathway of DEGs. (C) Network of GO biological processes of DEGs. (D) Five leading-edge gene sets using GSEA. (E) Heatmap of enriched genes for each gene set. S1, S2, and S3 represent three replicates treated with TBM I C1, C2, and C3 represent three replicates of PBS control. (F) PPI network of core genes from GSEA. (G) The Upset plot of 6 overlapping hub genes by seven algorithms.

Figure 8

The predicted target of adjuvant activity of TBM I. (A) Overlap of therapeutic targets of TBM I (green) and core genes from GSEA of microarray data (orange). (B) Enriched terms of overlapped targets. (C) Key targets and their PPI network. (D) The expression level of key targets in microarray assay. The data are expressed as means ± SD (n = 3). ^* P < 0.05, ^** P< 0.01, and ^*** P < 0.001 vs Ctrl. (E) Binding pattern diagram of TBM I and target protein SYK or LYN. The yellow dashed line represents the hydrogen bond interaction.

Figure 9

SYK-STAT3 pathway contributed the inflammatory response in C2C12 cells induced by TBM I. (A) The gene expression of IL-6 and PTGS2 in C2C12 cells induced by TBM I with RT-qPCR. (B) C2C12 cells were pretreated with SYK inhibitor (R406, 8 µM, 1 h), LYN inhibitor (SU6656, 2 μM, 1 h) or STAT3 inhibitor (S3I-201, 100 μM, 1 h) before TBM I (40 µM) stimulation for 4 h. The gene expression levels of IL-6 and PTGS2 in C2C12 cells were determined by RT-qPCR. (C) After pre-incubation with or without R406 (8 µM, 1 h) or S3I-201 (100 μM, 1 h), C2C12 cells were treated with medium or TBM I (40 μM) for 12 h. The level of IL-6 in the culture supernatants of C2C12 cells by ELISA. (D) C2C12 cells were treated with TBM I (40 μM) for 0, 0.5, 1, and 2 h, and the protein levels of P-Syk/SyK levels were detected by Western blotting. (E), (F) After pre-incubation with or without R406 (8 µM, 1 h), C2C12 cells were treated with medium or TBM I (40 μM) for 4 h or 8 h, and the protein levels of P-Stat3/Stat3 (E) and COX-2 (F) were detected by Western blotting. The figure shown was representative of three independent experiments. Data were presented as mean ± SD (n = 3). ^a P < 0.05, ^b P < 0.01, and ^c P < 0.001. (G) Hypothetical pathway involving in the inflammatory response in C2C12 cells induced by TBM I.