| Objective Natural killer T (NKT) cells are a unique subset of the mature T lymphocytes that can recognize glycolipids presented by the nonclassical MHC class I molecule CD1d, as well as co-express the surface markers of NK cell and T cell. After activation viaα-galactosylceramide (α-GalCer), which is derived from a marine sponge and is well defined as an exogenous ligand of NKT cells, or an endogenous ligand, isoglobotrihexosylcera-mide (iGb3), NKT cells promptly secrete large amounts of both T helper 1 (Th1) cytokines ( such as IFN-γ), and T helper 2 (Th2) cytokines (such as interleukin (IL)-4 and interleukin (IL)-13), thereby exhibit multiple immunothe raputic effect in cancer, infectious diseases and autoimmune diseases (for example, multiple sclerosis, autoimmune diabetes and experimental autoimmune encephalomyelitis). However,the different cytokines produced by NKT cells after activation might have opposite effect,as Th1 cytokines often antagonize the action of Th2 cytokines and vice versa,which largely limits the clinic usage ofα-GalCer and iGb3.In resent years, many researchers try to design, synthesize or make modification onα-GalCer to search for new NKT cell agonists that may have superior properties for the treatment of autoimmune and inflammatory diseases. The glycoceramide OCH, which is a derivative ofα-GalCer with a substantially shorter sphingosine chain, can stimulate NKT cells to preferentially produce Th2 cytokines, thereby indicating a potential application in the treatment of autoimmune conditions. Anotherα-GalCer analogue,α-C-GalCer, exerts more potent capacity to activate NKT cells and acts as a more effective trigger for IL-12 and IFN-γproduction, and thus is more valuable for resistance to tumor and infections. It was implication that modification of the ceramide chains could polarize the secretion of Th1 and Th2 cytokines by NKT cells. Although there are multitude investigations about the structure–activity relationship of modifiedα-GalCer, the exploration for chemically-modified iGb3 is absent.In this regard, we synthesized some iGb3 analogues by chemcal synthetic methods and have found that two iGb3 analogues, 4-HO-iGb3 and 4?-dh-iGb3, can increases IFN-γproduction by hepatic NKT cells. In this study, tetramer staining technology was used to further identify their effect. To explore the molecular mechanism of their preferrential Th1-baised cytokine secretion capacity by NKT cells, the structure of the CD1d/glycolipid/TCR complex was analyzed by QSAR software, and expression and activation of some transcription factors involving in Th1/Th2 differenation were examined. This research will provide a new strategy and the theoretical basis for the designation and clinical application of glycolipids.Methods(1) Murine hepatic and splenic NKT cell number and intracellular cytokines IFN-γand IL-4 expression, as well as the phosphorlation level of transcription factors STAT1and STAT6 were analyzed by flow cytometry.(2) Dendritic cells were induced from mouse bone marrow in vitro.(3) The level of IFN-γand IL-4 in serum and the culture supernatants was determined by ELISA method.(4) The gene expression of IFN-γ, IL-4, GATA-3 and T-bet was detected by real-time quantitative PCR.(5) The affinity and stability between glycolipid and TCR, or glycolipid and CD1d were analyzed by QSAR softwar.(6) The T-bet and GATA-3 expression in protein level was detected by Western blot. Results1. Chemical modified iGb3 analogue 4'''-dh-iGb3 preferentially increased IFN-γproduction by hepatic and splenic NKT cells.Firstly, C57BL/6 mice were treated with iGb3 and its analogues intraperitoneally, we found that, compared with iGb3, chemical modified iGb3 analogues 4'''-dh-iGb3 did not increase hepatic and splenic NKT cells in proportion and quantity, but significantly enhanced IFN-γsecretion in serum, and up-regulated the levels of IFN-γ+NKT cells of liver and spleen, while the expression and secretion of Th2 type cytokine IL-4 were not influenced significantly. Similar results were obtained by murine NKT cell lines stimulated with bone marrow-derived DC loading glycolipids in vitro.2. 4'''-dh-iGb3 increased the affinity between glycolipid and CD1d, and enhanced the stability of binding between glycolipidand TCR.The spatial structure and docking between glycolipid, TCR and CD1d was established on computer by QSAR software. Our results revealed that the hydroxy group introduced in the phytosphingosine of iGb3, as well as the original hydroxy group in the acyl chain, inserted into antigen-binding hydrophobic pocket of CD1d molecules directly, and thus enhanced the affinity between glycolipid and amino acids Asp80 (C) of CD1d molecules. Removing the 4'''hydroxyl group of the terminal galactose of iGb3, carried amino acid Lys167 of TCR from this side to the other side of glycolipid in the spatial location, thus increased the combination score between glycolipid and TCR from 3.3 to 4.9. Therefore, the binding affinity between 4'''-dh-iGb3 and TCR of NKT cells was enhanced significantly. These results indicated that 4'''-dh-iGb3 has hiher binding affinity to CD1d of antigen-presenting cells, andstronger stability of binding to TCR of NKT cell than iGb3.3. 4'''-dh-iGb3 up-regulated Th1-type cytokines-directed transcription factor STAT1 phosphorylation and T-bet expression.C57BL/6 mice were injected with iGb3 and its analogues intraperitoneally, or mouse NKT cell lines were stimulated in vitro. The expression levels of transcription factors STAT1 and T-bet were examined. The results showed that, compared with iGb3, 4'''-dh-iGb3 stimulation increased the level of STAT1 phosphorylation and T-bet expresssion, while the Th2 type cytokines associated transcription factor STAT6 phosphorylation and GATA-3 expression were not changed significantly.Conclusions(1) iGb3 analogue 4'''-dh-iGb3 stimulates Th1-baised immune responses of mouse NKT cells.(2) 4'''-dh-iGb3 increases the stability and affinity between CD1d glycolipid/NKT TCR complex, so that promotes NKT cell activation and Th1-type cytokine secretion.(3) 4'''-dh-iGb3 up-regulates Th1-type cytokines associated transcription factor STAT1 phosphorylation and T-bet expression.(4) 4'''-dh-iGb3 could be used as a new immune stimulator for treatment of cancer and infectious diseases.
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