Fig. 2.1
Schematic model illustrating the signaling pathways involved in macrophage activation by Lycium barbarum polysaccharides. L. barbarum polysaccharides can activate macrophages via Toll-like receptor 4 (TLR4) and TLR2. TLR4/2-activated signaling pathways lead to activation of phosphoinositide-3-kinase (PI3K) and LKB1, leading to activation of the mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB), p53, C-Jun, and AP-1. Ultimately, these pathways lead to induction of gene transcription. TLR4 ligation leads to the activation of IL-1R-associated kinase (IRAK) via adaptor myeloid differentiation protein 88 (MyD88), with subsequent activation of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF-6), MAP kinases (e.g., p38 and JNK) and NF-κB. Activation of these transcription pathways induces expression of pro-inflammatory cytokines and immune regulation, survival and proliferation
Table 2.1
Effect of L. barbarum on immune target cell
2.3.1 Target Cells
2.3.1.1 Dendritic Cells
Dendritic cells (DCs) represent a heterogeneous population of antigen-presenting cells that initiate the primary immune response (Banchereau et al. 2000). These cells take up antigens in peripheral tissues and migrate to secondary lymphoid organs where they become mature and competent in presenting antigens to T cells, thus initiating antigen-specific immune responses or immunological tolerance (Guermonprez et al. 2002). DC immunogenicity correlates with the DC functionally mature state, which is characterized by high-level expression of MHC and T cell costimulatory molecules, acute decreases in antigen uptake, and the ability to present antigens captured in the periphery to T cells (Wilson and Villadangos 2005). DC maturation can be induced by microbial products (such as LPS) or inflammatory cytokines (such as TNF) (Winzler et al. 1997). Although these mediators are potent stimuli of DC maturation, they are toxic and have limited applications. In this regard, as biological response modifiers (BRMs), polysaccharides are able to induce DC maturation and immunogenicity.
LBPs are known to exhibit immunomodulatory functions, including activation of B cells and natural killer (NK) cells. However, little is known about the immunomodulatory effects of LBPs on DC. The effects of LBPs on the phenotypic and functional maturation of murine BMDC were investigated in vitro. Compared with BMDC in the control group that was exposed to RPMI 1640 only, the co-expression of I-A/I-E, CD11c, and secretion of IL-12 p40 from BMDC were increased by LBPs (100 µg/ml). In addition, the endocytosis of FITC-dextran by LBPs-treated BMDC (100 µg/ml) was impaired, whereas the activation of proliferation of allogenic lymphocytes by BMDC was enhanced. The results strongly suggest that LBPs are capable of promoting both the phenotypic and functional maturation of murine BMDC (Zhu et al. 2007). Both LBPs and polysaccharide-protein complex isolated from L. barbarum can induce phenotypic and functional maturation of DCs with strong immunogenicity. Research has demonstrated that LBPs upregulate DC expression of CD40, CD80, CD86, and MHC class II molecules; down-regulate DC uptake of antigens; enhance DC co-stimulatory activity; and induce IL-12p40 and p70 production. Of all five fractions, LBPF1–5 has been demonstrated to be active. L. barbarum polysaccharide-protein complex enhances Th1 responses, and polysaccharide-protein complex-treated DCs enhance Th1 and Th2 responses in vitro and in vivo. The research provides evidence and a rationale for using L. barbarum in the treatment of various clinical conditions to enhance host immunity and suggests L. barbarum is a potent adjuvant in the design of DC-based vaccines (Chen et al. 2009a).
2.3.1.2 Macrophages
Macrophages play a major role in the host defense against infection. Macrophages express a broad range of pattern recognition receptors (PRRs) to bind the conserved structures of pathogens, ingest bond microbes into vesicles, and produce reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (mainly nitric oxide) to destroy microbes (Aderem and Underhill 1999). Activated macrophages also secrete the cytokines TNF-α and IL-1 and chemokines to induce inflammatory reactions to microbes (Pylkkanen et al. 2004). In addition, macrophages can present antigen to T cells and produce IL-12 to coordinate innate and adaptive immune responses (Watford et al. 2003). Furthermore, macrophages are involved in tissue remodeling after infections and injury, clearance of apoptotic cells and hematopoiesis (Krysko et al. 2006).
LBPF4-OL is the glycan part of L. barbarum polysaccharide-protein complex fraction 4 (LBPF4). A study demonstrated that the LBPF4-OL markedly induced proliferation of spleen cell but could not induce proliferation of purified T and B lymphocytes. Further study revealed that the proliferation of B cell took place in the presence of activated macrophages or LPS. Multiplex bead analysis indicated that LBPF4-OL can obviously induce production of IL-6, IL-8, IL-10 and IFN-α by spleen cells in a concentration-dependent manner. Flow cytometric analysis indicated that LBPF4-OL (i.p.) triggers expression of CD86 and MHC-II on macrophages. An enzyme-linked immunosorbent assay (ELISA) assay demonstrated that LBPF4-OL could greatly stimulate macrophages to secrete TNF-α and IL-1β. These results suggest that the glycan LBPF4-OL plays an important role in the immunopharmacological activity of L. barbarum polysaccharide-protein complex; and macrophages, rather than T and B cells, are the principal target cells of LBPF4-OL (Zhang et al. 2011).
It has been found that polysaccharide-protein complex from L. barbarum fruit (50 mg/kg, i.p.) markedly upregulated the expressions of CD40, CD80, CD86, and MHC class II molecules on peritoneal macrophages. In vitro studies demonstrated that L. barbarum polysaccharide-protein complex activated transcription factors NF-κB and AP-1 in RAW264.7 macrophages; induced mRNA expression for TNF-α, IL-1ß, IL-12p40; and enhanced production of TNF-α in a dose-dependent manner. Furthermore, L. barbarum polysaccharide-protein complex (50 mg/kg, i.p.) significantly enhances endocytic and phagocytic capacities of macrophages in an in vivo study. These results indicate that L. barbarum polysaccharide-protein complex enhances innate immunity by activating macrophages. The mechanism may be mediated via activation of transcription factors NF-κB and AP-1 to induce production of TNF-α and upregulation of MHC class II (Chen et al. 2009b). The comparisons of immune activities of polysaccharides and polysaccharide-protein complex from L. barbarum on macrophages have also been reported. Experiments using in vitro assays found that LBPF4-induced proliferation of splenocytes was dependent on both B and T cells. However, LBPF4-OL-induced splenocyte proliferation was mainly dependent on B cells. The ELISA results indicated that both LBPF4 and LBPF4-OL significantly induced production of TNF-α, IL-1ß, and NO from macrophages. Furthermore, electrophoretic mobility shift assay (EMSA) studies suggest that LBPF4 100 µg/ml can be more effectively to increase NF-kappa B activity than that of LBPF4-OL. The results demonstrate that LBPF4 can enhance T, B cells, and macrophage functions, but LBPF4-OL can only enhance B cells, and macrophage functions. This is partly due to LBPF4 being able to more significantly enhance lymphocytes NF-κB activity (Zhang et al. 2014a).
2.3.1.3 T Lymphocytes
The present research has revealed the effects of L. barbarum in activating T cells. Flow cytometry assays revealed that L. barbarum polysaccharide enhanced the proliferation of murine splenic lymphocyte. The combined use of LBP and Con A had synergistic effects. MTT assays demonstrated that LBP significantly promoted proliferation of murine splenic lymphocytes, whereas LBP plus Con A combination also enhanced the lymphocyte proliferation at a high concentration. LBP with Con A had effects on immunocompetence (Amagase and Farnsworth 2011). Another research group found that LBP significantly stimulated proliferation of mouse splenocytes. T cell but not B cell proliferation was observed. Cell cycle profile analysis indicated that LBP5 markedly reduced sub-G1 cell expression. LBP could activate the transcription factors NFAT and AP-1, prompt CD25 expression, and induce IL-2 and IFN-γ gene transcription and protein secretion. LBP (i.p. or p.o.) significantly induced proliferation of T cells. The effect of L. barbarum glycopeptide 3 (LBGP3) on T cell apoptosis in aged mice has been reported. LBGP3 was purified from Fructus Lycii water extracts and identified as a 41 kD glycopeptide. Treatment with 200 µg/ml LBGP3 increased the apoptotic rate of T cells from aged mice and produced a similar DNA ladder pattern to that observed in young T cells. The reversal of apoptotic resistance was involved in down-regulating the expression of Bcl-2 and FLIP and upregulating the expression of FasL. L. barbarum glycopeptide 3 reverses apoptotic resistance of aged T cells by modulating the expression of apoptosis-related molecules (Yuan et al. 2008). The results suggest that activation of T lymphocytes by L. barbarum may contribute to one of its immune enhancement functions (Chen et al. 2008a).
2.3.1.4 NK Cells
Polysaccharides are believed to be strong immune stimulants that can promote the proliferation and activity of T cells, B cells, macrophages, and NK cells. A study aimed to investigate the effects of polysaccharides, including L. barbarum polysaccharide (LBP), on primary human NK cells under normal or simulated microgravity (SMG) conditions. The results demonstrated that LBP markedly promoted the cytotoxicity of NK cells by enhancing the secretion of IFN-γ and perforin and increasing the expression of the activating receptor NKp30 under normal conditions. Moreover, LBP can enhance NK cell function under SMG conditions by restoring the expression of the activating receptor NKG2D and reducing early apoptosis and late apoptosis/necrosis. Additionally, antibody neutralization tests demonstrated that CR3 may be the critical receptor involved in polysaccharide-induced NK cell activation. These findings indicate that polysaccharides may be used as immune regulators to promote the health of the public and even astronauts during space missions (Ting et al. 2014).
2.3.1.5 Other Target Cells
Granular leukocytes and mast cells are the main effector cells of food allergies, which cause type ǀ hypersensitivity. L. barbarum berries have been introduced into Western diets. Preliminary reports have demonstrated its allergenic capacity (Ballarin et al. 2011; Larramendi et al. 2012). A study investigated the frequency of sensitization and the allergens. In this study, 566 individuals with respiratory or cutaneous symptoms were skin prick tested with L. barbarum berry extract. Thirty-three individuals were positive (5.8 %), and 94 % were sensitized to other allergens. The specific IgE to L. barbarum berries, peaches, tomatoes, and a nut mix was measured. Thirteen individual serum samples out of 24 available serum samples (54.2 %) had positive specific IgE. In addition, 92.3 % of L. barbarum berry-positive patients were positive to peaches. Seven individuals recognized eight bands, and six recognized a 7-kDa band. This band was identified as a lipid transfer protein by mass spectrometry (MS/MS). Cross-reactivity was demonstrated with tomato, tobacco, nut mix, Artemisia pollen, and purified Lyce3 and Pru p3. These results indicate that L. barbarum berries are a new allergenic source with a high prevalence of sensitization (Carnes et al. 2013). Some other reports found that the isolated active component of LBP3a, combined with a DNA vaccine encoding the major outer membrane protein of Chlamydophila abortus, induced protection in mice against challenge. A combination of DNA vaccine and LBP3a induced significantly higher antibody levels in mice. MOMP-specific IgG1, IgG2a, and IgG2b antibodies were found in the pool of sera postvaccination on day 42. IgG2a and Ig2b became the predominant isotypes in 12.5, 25, and 50 mg/kg LBP3a-adjuvanted groups (Ling et al. 2011). It has also been reported that mice fed L. barbarum had higher influenza antibody titers (Du et al. 2014). These studies indicate that granular leukocytes, mast cells, and B lymphocytes are also related to the L. barbarum activity. However, more experiments are required to clarify whether these cells are the direct target cells of L. barbarum.
2.3.2 Receptors
Identifying cellular receptors is important to understand how polysaccharides exert their immunomodulatory effects. Several ß-glucan receptors have been identified. The reported ß-glucan receptors include lactosylceramide (LacCer), Toll-like receptors (TLRs) 2 and 6, and dectin-1 (Zimmerman et al. 1998; Sletmoen and Stokke 2008). In addition, TLR4 has been identified as a receptor of polysaccharides, and many polysaccharide activities involve TLR4. Other polysaccharide-related receptors have been reported, including complement receptor-3 (CR3), scavenger receptor (SR), MR (CD206), CD44/RHAMM and selectins.
A study reported that the activity of the polysaccharide LBPF4-OL, which was purified from LBP, is closely associated with the TLR4-MAPK signaling pathway. Research found that LBPF4-OL could significantly induce production of TNF-alpha and IL-1ß in peritoneal macrophages isolated from wild-type (C3H/HeN) but not TLR4-deficient mice (C3H/HeJ). The study also found that the proliferation of LBPF4-OL-stimulated lymphocytes from C3H/HeJ mice is significantly weaker than that of lymphocytes from C3H/HeN mice. Furthermore, through a bio-layer interferometry assay, it was found that LPS but not LBPF4-OL can directly associate with the TLR4/MD2 molecular complex. Flow cytometry analysis indicated that LBPF4-OL markedly upregulates TLR4/MD2 expression in both peritoneal macrophages and Raw264.7 cells. As its mechanism of action, LBPF4-OL increases the phosphorylation of p38-MAPK and inhibits the phosphorylation of JNK and ERK1/2, as was examined by western blot analysis. These data suggest that the L. barbarum polysaccharide LBPF4-OL is a new Toll-like receptor 4/MD2-MAPK signaling pathway activator and inducer (Zhang et al. 2014b). Similar results have been observed for dendritic cells. Zhu et al. reported that LBPs induced phenotypic and functional maturation of DCs. LBPs upregulated DC expression of I-A/I-E and CD11c, enhanced DC allostimulatory activity and induced production of IL-12p40. Furthermore, the activity of LBPs on DCs was significantly reduced by treating the cells with anti-TLR2 or anti-TLR4 antibody prior to LBPs, indicating that both are possible receptors of LBPs. Maturation of DCs by LBPs was able to directly activate the nuclear transcription factor NF-κB p65. The results revealed that LBP stimulation induces the phenotypic and functional maturation of DCs via TLR2 and/or TLR4-mediated NF-κB signaling pathways (Zhu et al. 2013). The above results indicate the immunoactivity of L. barbarum is related to TLR4/2. Whether other receptors are related to L. barbarum remains unknown.
2.3.3 Signal Transduction
Several signal transduction pathways, including the PI3K/Akt/FoxO1, LKB1/AMPK, JNK/c-Jun, MEK/ERK, and PI3K/HIF-1α pathways, and transcription factors NF-κB, p53, c-Jun, and AP-1 are reported to be L. barbarum activity-related signal transduction molecules.
L. barbarum polysaccharides (LBPs) from wolfberries have been reported to have antioxidant and neuroprotective derivatives. A study found that LBPs are also a novel hepatoprotective agent against nonalcoholic steatohepatitis (NASH) caused by a diet-induced NASH rat model. The study examined female rats fed with 1 mg/kg LBP daily for 8 weeks and compared with control rats. NASH+ LBPs-cotreated rats displayed (1) improved histology and free fatty acid levels, (2) re-balancing of lipid metabolism, (3) reducing profibrogenic factors through the TGF-ß/SMAD pathway, (4) improved oxidative stress through the cytochrome P450 2E1-dependent pathway, (5) reducing production of hepatic pro-inflammatory mediators and chemokines, and (6) ameliorating hepatic apoptosis through the p53-dependent intrinsic and extrinsic pathways. All these effects of LBP were partly modulated through the PI3K/Akt/FoxO1, LKB1/AMPK, JNK/c-Jun, and MEK/ERK pathways and the down-regulation of transcription factors in the liver, such as NF-κB and activator protein-1 (AP-1) (Xiao et al. 2013). Moreover, LBPs have also been found to inhibit tumor cell growth by suppressing IGF-1-induced angiogenesis via PI3K/HIF-1α/VEGF signaling pathways. Studies have reported that a 90 h treatment with 0.50 mg/ml of LBPs resulted in significant inhibition of MCF-7 cell proliferation. Using this same cell type, studies have also observed that LBPs could also affect insulin-like growth factor (IGF)-1 protein accumulation, suppress PI3K activity and p-PI3K protein levels, inhibit accumulation of hypoxia-inducible factor-1 (HIF-1α) protein without altering HIF-1α mRNA levels, and suppress mRNA expression and protein production of VEGF (Huang et al. 2012).
NF-κB has also been found to be one of the most important transcription factors related to L. barbarum activity. A study reported that LBP treatment may protect against intestinal ischemia-reperfusion injury (IRI)-induced intestinal damage by inhibiting PMN accumulation and ICAM-1 expression and ameliorating changes in TNF-α level, NF-κB activation, intestinal permeability, and histology (Yang et al. 2013). Other reports indicate that LBPs do not delay primary degeneration of RGCs after either complete optic nerve transection (CONT) or partial optic nerve transection (PONT), but they delay secondary degeneration of retinal ganglion cells (RGCs) after PONT. The study found that LBPs appeared to exert these protective effects by inhibiting oxidative stress and the JNK/c-Jun pathway and by transiently increasing production of insulin-like growth factor-1 (IGF-1) (Li et al. 2013). After investigating the effect of LBP on the differentiation and maturation of healthy human peripheral blood-derived dendritic cells cultured in different tumor microenvironments in vitro and evaluating the molecular and immunological mechanisms of LBP in the treatment of tumors, a study reported that LBPs could increase the expression of the phenotype of DCs, secretion of IL-12p70 and IFN-γ in MLR and enhance NF-κB expression, especially in virus-related peripheral blood-derived dendritic cell precursor cells, suggesting that LBPs play a stronger antitumor role in virus-related environments, and this phenomenon correlates with the NF-κB signaling pathway (Chen et al. 2012). Maturation of DCs by LBPs is able to directly activate the nuclear transcription factor NF-κB p65 (Zhu et al. 2013).
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