Study On The Structure-activity Relationship And The Mechanism Of Multi-pathway Synergistic Immune Response Of Glucomannans Based On The Recognition Of Innate Immune Receptors

In recent years,frequent evidence of interaction between different structural polysaccharides and Toll-like receptor（TLR）or C-type lectin receptor（CLR）has pushed the theory that polysaccharides are involved in the regulation of the innate immune system through innate immune receptors to the forefront of research.However,the patterns and mechanisms of interaction between polysaccharides and innate immune receptors are still unclear,such as why TLR4,the“star”interacting receptor of polysaccharides,can recognize multiple structural types of polysaccharides.Therefore,based on the“non-self”theory of innate immune receptor recognition and the literature overview,this thesis condenses the hypothesis that“Innate immune receptor co-recognition of glucomannans initiates multi-pathway synergistic immune response”and selected three different structural glucomannans（Konjac glucomannan（KGM）,Aloe vera gel polysaccharide（AGP）,Dendrobium officinale polysaccharide（DOP））were selected to test the authenticity and generalizability of the hypothesis.Finally,the application of this hypothesis provides some new insights into the structure-activity relationship between the acetyl group of glucomannans and macrophage immune activation.The main contents and results are summarized as follows:（1）A systemic immunosuppression model based on cyclophosphamide（CTX）was used to further characterize the in vivo immunoregulatory activity of glucomannans.The results showed that glucomannans alleviated the immunosuppression caused by CTX by enhancing the cellular and humoral immune functions.The enhancement of cellular immune function was demonstrated by increasing the total number of circulating lymphocytes in the blood,stimulating the secretion of various cytokines in the spleen（IFN-?secretion in KGM,AGP,and DOP groups was 9.9,5.4,and 9.4 times higher than that in the model group,respectively）,regulating the ratio of CD4⁺and CD8⁺T cell subsets in the spleen,and upregulating the expression of key nuclear transcription factor T-bet（up to 2.2,2.7,and 1.8 times of the model group,respectively）in Th1-type T cells.The stimulation of humoral immune function was manifested by the upregulation of plasma cell differentiation gene XBP-1（up to 1.6,2.1,and 2.0 times of the model group,respectively）to promote immunoglobulin production.The above results suggested that three glucomannans exhibit good in vivo immunomodulatory potential by enhancing cellular and humoral immune functions to different degrees in the immunosuppressive microenvironment.（2）The RAW264.7 macrophage cell line model was used to evaluate the macrophage activation potential of glucomannans in vitro.The results showed that glucomannans enhanced all immune functions of macrophages.20-80?g/ml of glucomannans significantly increased the proliferation,endocytosis,and uptake of antigens by macrophages.The secretion of immune products showed that glucomannans selectively promoted the secretion of various pro-inflammatory mediators by macrophages,such as the secretion of TNF-αin KGM,AGP,and DOP groups was 212.7,257.9,and 86.7 times higher than that in the model group,respectively.Glucomannans tended to increase the expression of molecular markers related to M1 phenotype,such as extracellular CD86 and intracellular Nos2 expression（269.8,226.3,and 111.2 more than the control group,respectively）.Classical NF-κB and MAPK pro-inflammatory signaling pathway protein expression revealed that glucomannans increased the degradation of IκB-αand phosphorylation of JNK,ERK,and p38 in the cytoplasm thereby inducing the expression of nuclear transcription factors NF-κB and c-Jun and their phosphorylation modifications（1.7,1.7,and 1.4-fold of control for p-NF-κB;4.7,10.5,and 7.1-fold of control for p-c-Jun,respectively）.The above results indicated that glucomannans comprehensively initiated immune activation in macrophages,which was manifested by promoting immune products secretion,enhancing endocytosis and phagocytosis,regulating immune phenotype,and activating pro-inflammatory signaling pathway transduction.（3）Based on macrophage cell line,the authenticity and generalizability of the hypothesis were confirmed for the first time at four levels:molecular expression,activity inhibition,gene knockdown,and molecular docking.The receptor inhibitor screen showed that TLR4 and MD2 activity deficiency significantly blocked the secretion of various inflammatory mediators by glucomannan-stimulated macrophages,such as TLR4 inhibitor reduced TNF-αsecretion in KGM,AGP,and DOP groups by428.5,323.1,and 225.1 times,respectively.Silencing TLR4,MD2,and CD14 genes significantly impaired the protein expression and phosphorylation levels of NF-κB and c-Jun elevated by glucomannans.Glucomannans also promoted the formation of TLR4-MD2 dimer on the cell surface,where AGP stimulation resulted in the formation of approximately 40%of the dimer within the first 30 min of stimulation.Besides,silencing of the mannose receptor（MR）also significantly blocked glucomannans from elevating the secretion of various inflammatory mediators（ROS secretion was reduced by 9.7,5.7,and 5.3-fold,respectively）and the phosphorylation level of NF-κB in the nucleus.Downstream analysis of key transduction molecules revealed that MR co-mediated glucomannan-activated NF-κB signaling pathway in a Syk-dependent manner in synergistic with TLR4 signaling pathway,while MR synergized with membrane surface clathrin to endocytose glucomannans into the cytoplasm to promote TNF-αsecretion and thereby synergistically amplify the inflammation-activated phenotype.The molecular docking results showed that the glucomannans repeat unit（Aloe oligosaccharide,oligo-AGP）exhibit optimal binding stability to TLR4-MD2 dimer,followed by the C-type lectin-like domain 4（CTLD4）binding stability to MR.The above results provided evidence one for hypothesis:in the immune microenvironment of a single macrophage cell line,glucomannans are recognized by the cell surface CD14-TLR4-MD2 recognition cascade in cooperation with MR and synergistically promotes intracellular multi-pathway signaling,such that glucomannans activates the classical NF-κB pro-inflammatory signaling pathway in a Syk-dependent manner in cooperation with TLR4/Myd88 through MR,and MR synergizes clathrin to internalize glucomannans to further enhance inflammatory activation.（4）A homologous transplantation tumor model was constructed based on the CT26 tumor cell line,and intratumoral injection was used to demonstrate that glucomannans directly initiates anti-tumor immune responses through multiple innate immune pathways in the complex tumor microenvironment.Intratumoral injection of KGM and AGP resulted in 37.7%and 41.8%reduction in tumor volume and 37.9%and 44.0%tumor inhibition,respectively,while DOP injection had little effect on tumor growth.Pathological analysis showed large areas of tumor cell necrosis and nuclei loss in the KGM and AGP injection groups.Immuno-infiltration analysis revealed that KGM and AGP significantly increased the proportion of M1-type macrophages（tumor suppressor phenotype）and significantly decreased the proportion of M2-type macrophages（tumor promoter phenotype）to initiate anti-tumor immune responses,and KGM and AGP also upregulated the proportion of CD8⁺T cells（from12.7%to 21.0%and 19.4%,respectively）.Besides,KGM and AGP reversed the tumor immunosuppressive microenvironment by promoting the secretion of tumor suppressor and decreasing the m RNA expression of tumor-promoting factor.RNA-seq immune signaling pathway mining revealed that the reversal of macrophage tumor-promoting phenotype by glucomannans was mediated by multiple innate immune receptors signaling pathways（TLRs,CLRs,and NLRs）and that the downstream key nuclear transcription factors were STAT1,IRF1,and IRF5.The above results provide evidence two for hypothesis:In the complex tumor microenvironment,KGM and AGP activate multiple key nuclear transcription factors（STAT1,IRF1,and IRF5）through multiple innate immune pathways（TLRs,CLRs,and NLRs）thereby initiating potent anti-tumor immune responses.（5）The in vitro construction of the bone marrow-derived macrophages（BMDM）model reconfirmed that the hypothesis also applies to glucomannan-induced differentiation of BMDM to M1 phenotype.The percentage of M1 macrophages induced by 320?g/m L KGM,AGP,and DOP was 89.8%,81.7%,and 83.4%,respectively（control group was only 40.2%）.Glucomannans also significantly upregulated the m RNA expression of tumor suppressor in M1 macrophages（IL-12 was upregulated to 174.2,97.5,and 27.2-fold of the control group,respectively）.Co-culture of BMDM with tumor cells revealed that glucomannan-induced phenotypic transformation of BMDM exhibits anti-tumor potential,as evidenced by a significant reduction in the proliferation viability of cancer cell and an enhanced ability of BMDM to present tumor antigens（F4/80⁺MHCII⁺ratios were 2.1,2.2,and 2.5 times higher than control group,respectively）.Gene silencing or activity inhibition repeatedly confirmed the involvement of TLR2,TLR4,NF-κB,JAK1,and STAT1 in glucomannan-induced differentiation of BMDM to M1 type.Among them,JAK1activity inhibition downregulated M1 type macrophages 3.2,3.1,and 3.5 times more than untreated group,respectively.The results of effector gene m RNA expression revealed that glucomannans cooperated with NF-κB and JAK1-STAT1 signaling pathway through TLR2 and TLR4 to regulate the m RNA expression of type I interferon and effector gene IRF1.The above results provided evidence three for hypothesis:In an in vitro BMDM induction model,glucomannans cooperates with NF-κB signaling pathway and JAK1-STAT1 signaling pathway through TLR2 and TLR4 to potently regulate BMDM phenotypic differentiation and effector functions,first insight into the mechanism by which glucomannans initiates the type I interferon autocrine loop through TLR2 and TLR4 to form a JAK1-STAT1 positive feedback loop.（6）Glucomannans was modified by deacetylation and acetylation using Na₂CO₃and acetic anhydride,and the structure-activity relationship between glucomannans acetyl and macrophage immune activation was elucidated based on the hypothesis.Differences in the degree of addition or removal of different glucomannans acetyl revealed inconsistencies in the rate and ease of reaction.Both acetylation and deacetylation resulted in a decrease in the solubility of the modified glucomannans and a significant change in the surface solid morphology.The immune activation assay revealed that the activation potential of modified glucomannans showed an increasing and then decreasing trend with the degree of acetyl substitution,revealing that there was not a linear positive or negative,but rather a“nonlinear wave”dose-effect relationship between them.Based on the hypothesis that the object of the structure-activity relationship is clearly explored for the relationship between acetyl groups and innate immune receptor binding,it can be briefly described as the acetyl group changes the conformation of the active chain of glucomannan-changes in the total number of hydrogen bond receptors and donors binding to active sites-changes of hydrogen bonding interactions with receptors-transformation of the“non-linear wave”immune activation phenotype.The docking binding energy（stability）between different acetyl substitution oligo-AGP and TLR4-MD2 monomer or CTLD4 also showed a“nonlinear wave”trend,and the number,site,and posture of interacting hydrogen bonds changed dramatically,providing potential evidence for the elaboration of structure-activity relationship.The above results reveal that the dose-effect relationship between glucomannans acetyl and macrophage activation phenotype is“nonlinear wave”,and the potential structure-activity relationship can be clarified by applying the hypothesis.