Molecular Mechanism Of Synthetized Glucohexaose Modulated The Innate Immune Response

Ganoderma lucidum and other edible Mushroom have been used for a long time in China to prevent and treat various diseases. Polysaccharides of fungal origin have important immunostimulatory effects, acting mainly in the activation of macrophages and dendritic cells through selective innate receptors (such as dectin-1). Recent pharmacodynamic characterisation of the active principles in such extracts has revealed that many polysaccharides and oligosaccharides are among the bioactive molecules. Among the best characterized bioactive molecules, polysaccharides has been shown to have potent anti-tumour activity due to its powerful capacity to stimulate cell mediated immune responses and to activate innate immune effector cells such as monocytes/macrophages and NK cells and DC. It also shows that polysaccharides have anti-inflammatory activity by promote the production of anti-inflammatory factor and inhibit the production of inflammatory factor. Besides for the anti-tumour, immunomodulator and anti-inflammatory capacity of polysaccharides, it shows protective effect on the autoimmune disease. Possibly because of its ability to stimulate immune response, polysaccharides, as an adjuvant, were found able to reduce tuberculosis infection and to enhance the effect of BCG vaccination and to enhance the immune response induced by HBV DNA vaccine. In addition, they can induce T cells immune response by T cell independent and T cell dependent way according to their structure. Indeed, the use of oligosaccharidic adjuvants appears very promising for targeted stimulation of innate immunity and could help to biasing the immune response towards more effective protection.β-(1â†'6)-branchedβ-(1â†'3) gluco-oligosaccharides are the common structural moiety of many bioactive polysaccharides from plants and fungi. The anti-tumour activity of lentinan, theβ-(1â†'3)-D-glycan with 2β-(1â†'6)-glucosyl branches every 5 residues, appears to be related to its structure and molecular weight. The triple-helix conformation of the backbone chain in lentinan is important for the biological effect, and the basic unitβ-(1â†'6)-branchedβ-(1â†'3) glucohexaose is believed to play a major role in anti-tumour activity. Indeed, theβ-(1â†'6)-branchedβ-(1â†'3)-glucoexahose and its analogues withα-(1â†'3)-linked bond show similar effects as lentinan in anti-tumour activity in the experimental S18 tumour model and in amplification of spleen cell proliferation and induction of TNF-a production. On the other hand, the P-glucan mainly containing 1,6 linkages is significantly less active. Thus, it appears that the basic unit ofβ-glucan is endowed with the immunostimulatory effects of the whole polysaccharide.With the similar effecter of polysaccharides, the simple structure, high purity and high yield of, synthetized glucohexaose attracts more attention on the synthesis and bioactivity of glucohexaose. Based on the structure unit of lentinan, theβ-(1â†'6) branchedβ-(1â†'3) glucohexaose analogue which containing anα-(1â†'3)-linked bond (named asβ-oligosaccharide in this paper) was sythetized by professor Gu group. The immunomodulatory activity on innate immune response ofβ-oligosaccharide was examined in vivo and vitro in this thesis.Part I Regulation mechanism ofβ-oligosaccharide on innate immune response in macrophage Macrophages not only serve as the primary defense barrier in the innate immune response, but also act as important accessory cells in the adaptive immune response. Macrophages were treated byβ-oligosaccharide (100μg/ml). The expression of activation related cell surface molecules was assessed by flow cytometry. MHC classâ… /â…¡and co-stimulating molecules (CD40, CD86) are related to antigen presenting capacity. The gene transcripation of M1/M2 cytokines was measured by quantitative real-time RT-PCR, TNF-α, IL-12, IL-1βand IL-6 mRNA were measured as M1-related inflammatory cytokines, while IL-10, IL-1Ra were assessed as M2 activation cytokines. The secretion of TNF-α, IL-6, IL-12 and IL-10 was measured by ELISA assay. The results indicated thatβ-oligosaccharide has up-regulating the gene transcripation of M1 cytokines, the secretion of TNF-αand IL-12, and the expression of activation related cell surface molecules. To examine the possible involvement of TLR2, TLR4 and Dectin-1 inβ-oligosaccharide induced TNF-α, IL-6, IL-12 and IL-10 secretion from macrophages, the specific blocking antibodies were used. As results shown, TLR2, TLR4 and Dectin-1 have relationship with the function ofβ-oligosaccharide on modulating secretion of cytokines.Immunoregulation is a network including immune cells, cytokines and signaling pathways. To investigate the regulatory effect ofβ-oligosaccharide on immune response pattern of mice immune cells, macrophages were treated byβ-oligosaccharide (100μg/ml) for 4 hours, and the differentially expressed genes were screened by qPCR array.38 differentially expressed genes were identified, of which 18 genes were up-regulated and 20 genes were down-regulated. They are mainly genes of cytokines and cytokine receptors, including chemokines and chemokine receptors, interleukins and interleukin receptors, TNF superfamily and others. To analysis the differentially expressed genes, the transcription factors (TF) were predicted using a bioinformatics method. The sequence corresponding to the TF binding site (TFBS) was searched in the upstream region from the transcription starting site (TSS) of the genes, then TFs were matched with the corresponding TFBS, and TF genes were mapped to the signaling pathways.39 TFs were identified, of which 7 TFs (18%) can be mapped to MAPK signaling pathway. To investigate the mechanisms ofβ-oligosaccharide regulating macrophage activation, phosphorylation level of ERK, the key protein kinase of MAPK pathway (M1 polarization), and AKT, the key protein kinase of PI3K pathway (M2 polarization), were analyzed by Western-Blot analysis. We discovered thatβ-oligosaccharide has enhanced activity on ERK and inhibited activity on AKT activation. TLR2 is related to the function of P-oligosaccharide enhancing activity on ERK, and Dectin-1 is related to the effect of P-oligosaccharide on ERK and AKT activation. The secretion of chemotactic factors and cytokines was measured by cytokines array,β-oligosaccharide up-regulated the secretion of I CCL1, CCL2, CCL3, CCL4, CCL5, CCL12, CXCL10, TIMP-1, G-CSF, KC, CD54, MIP-2, IL-1α, IL-1β, IL-6,IL-16, IL-17, IL-23, IL-1ra, IFN-y and TNF-α.Part II Effects of P-oligosaccharide on the transcription of immune-related cytokines in HT29 cells, human PBMC/PBM and identification of signaling pathways in human PBMC exposed toβ-oligosaccharideHT29 cells and PBM were treated byβ-oligosaccharide (100μg/ml) respectively. The gene transcription of pro-inflammatory and anti-inflammatory cytokines (IL-18/IL-18BP) was measured by quantitative real-time RT-PCR. The results indicated that P-oligosaccharide has inhibited activity on the function of pro-inflammatory cytokine IL-18 in PBM:down-regulating the gene transcription of IL-18 and its receptors, up-regulating the mRNA level of IL-18 inhibitory factor IL-18BP, so increased the ratio of IL-18BP/IL-18.Immunoregulation is a network including immune cells, cytokines and signaling pathways. To investigate the regulatory effect of P-oligosaccharide on immune response pattern of human immune cells, PBMC were treated by P-oligosaccharide (100μg/ml) for 4 hours, and the differentially expressed genes were screened by qPCR array.22 differentially expressed genes were identified, of which 17 genes were up-regulated and 5 genes were down-regulated. They are mainly genes of cytokines and cytokine receptors, including chemokines and chemokine receptors, interleukins and interleukin receptors, TNF superfamily and others. To analysis the differentially expressed genes, the transcription factors (TF) were predicted using a bioinformatics method. The sequence corresponding to the TF binding site (TFBS) was searched in the upstream region from the transcription starting site (TSS) of the genes, then TFs were matched with the corresponding TFBS, and TF genes were mapped to the signaling pathways.38 TFs were identified, of which 7 TFs (18%) can be mapped to MAPK signaling pathway. To investigate the mechanisms ofβ-oligosaccharide regulating PBMC, activation phosphorylation level of ERK, the key protein kinase of MAPK pathway (M1 polarization) was analyzed by Western-Blot analysis. We discovered thatβ-oligosaccharide has enhanced activity on ERK activation. Human peripheral blood mononuclear cells (PBMC) are a group of blood cells having a round nucleus, including monocyte, NK cells, T cells and B cells, etc. Peripheral blood monocytes (PBM) are precursor of macrophages and of importance in inflammatory response. Monocytes were separated from PBMC by magnetic beads sorting. PBMC and PBM were treated by P-oligosaccharide (100μg/ml) respectively. The secretion of chemotactic factors and cytokines was measured by cytokines array. In PBMCs,β-oligosaccharide enhanced the secretion of CCL5, CXCL1, MCP-1, MIF, MIP-1 alpha and IL-6. In PBMs,β-oligosaccharide up-regulated the production of chemokines such as CCL5, CXCL1, MCP-1, MIF, MIP-1αand IL-8, and cytokines such as IL-6, TNF-a and IL-Ira.Part III Effect ofβ-oligosaccharide on the innate immune response induced by DNA vaccine encoding hepatitis B virus core antigenAccording to the reports, marrow dendritci cells (MDC) were differentiated from bone marrow cell with stimulation of IL-4 and GM-CSF and treated with 100ug/mLβ-oligosaccharide in vitro. The expression of co-stimulate molecular were detected by flow cytometry and results showed thatβ-oligosaccharide enhanced the expression of CD40, CD86, MHC I and MHC II (136.1,30.1,110.1 and 8.1 respectively; vs 123.4,20.6,60.6 and 3.42, mock, respectively). The gene transcripation of M1/M2 cytokines was measured by quantitative real-time RT-PCR, TNF-a, IL-12, and IL-6 mRNA were measured as M1-related inflammatory cytokines, while IL-10 was assessed as M2 activation cytokines. The secretion of TNF-a, IL-6, IL-12 and IL-10 was measured by ELISA assay. The results indicated thatβ-oligosaccharide up-regulats the gene transcripation of TNF-a and IL-12, the secretion of TNF-a and IL-6, while the gene transcription and secretion of IL-10 were down-regulated. It showed that P-oligosaccharide enhanced the maturation of MDC and modulated the secretion of cytokines. Being inducing antigen-specific cytotoxic T lymphocytes, DNA vaccine had been investigate extensively in protection and therapy of disease. We have observed the effect ofβ-oligosaccharide on the specific immune response induced by DNA vaccine encoding HBV core antigen (pB144). In this part, we evaluated the effect of P-oligosaccharide on the maturation of spleen dendritic cells induced by DNA vaccine encoding hepatitis B virus core antigen. Mice were immunized with P-oligosaccharide and pB144 and the immune response was measured at day 5 after immunization. Maturation of recruited DC was more pronounced in spleens of mice immunized with pB144 together with P-oligosaccharide, as compared to cells from animals receiving pB144 alone.