The Effects Of Lentinan On The T Cell Subsets In Intestinal Mucosal Immune System Of Mice

Lentinan (LNT) has a wide range of biological activities, including immune regulation, anti-tumor, anti-inflammation, antivirus, and so on. The immunomodulatory effects of which LNT is an important basis for their biological activity. Studies have shown that the LNT is a typical T-cell activator, which can induce macrophages to produce a variety of biologically active factor in vivo, promote T cell activation and proliferation, and enhance the function of Th and Tc cells, leading to the occurrence of a series of immune responses.LNT has been widely used to be the adjuvant therapies in the clinical for anti-tumor, anti-inflammatory treatments, but also been developed for a variety of natural health products. However, as polysaccharide is difficult to be absorbed by mammals, it’s still not clear that how can the polysaccharides regulate the immune system became the focus of studies.In recent years, the studies of intestinal mucosal immunity have been increasingly important, since he intestinal mucosal immune system (IMIS) is the largest and the most complex part of the immune system. Not only does it encounter more antigen than any other part of the body, but it must also discriminate clearly between invasive organisms and harmless antigens, such as food proteins and commensal bacteria. And IMIS can direct lymphocyte homing and recirculation to affect the peripheral immune system. IMIS may be the essential place that oral LNT encountered. Previous studies have shown that LNT can significantly improve the murine intestinal mucosal immune suppression and dysfunction, including enhance the sensitivity of T cells and recover the normal proportion of the lymphocyte subsets.This paper will choose Lentinan to explore the role of oral polysaccharides on the T cell subsets of intestinal mucosal immune system. To some extent, this study may be help us to uncover some of the immunomodulatory mechanisms of oral polysaccharides. Objective:To establish and optimize the detection of lymphocytes intracellular cytokines by flow cytometry. On the basis of delayed-type hypersensitivity (DTH) mice and immune-suppressive mice induced by cyclophosphamide (CY), this paper compares the effects of LNT on the T cell subsets in different tissues. Thus, to find the potential roles of LNT in the gut mucosal immunity.Methods:1. Optimization of the detection of intracellular cytokines by flow cytometry.Establish the method of detection of cytokines in mice lymphocytes by the evaluation of CD3/CD69proportion, optimize the experimental conditions, including the stimuli concentration and the better protein transport inhibitor. Lymphocytes were stimulated with different concentrations (25,50,75ng/ml) of PMA and1μg/ml Ionomycin for5h to determine the optimal concentration of PMA; The T cell surface and intracellular CD69was monitored in order to measure the efficacy of the two protein transport inhbitors (BFA/Monensin Mixture and Monensin).2. DTH mice model and the effects of LNT changes in different parts of the DTH mouse T cell subsets2,4-dinitrofluorobenzene(DNFB)-induced delayed type hypersensitivity (DTH) mice model was made. And the expression of T cell activation marker CD69of the intestinal inductive site (PPs), mesenteric lymph nodes (MLN), spleen (SP) and the retroauricular lymph nodes (RLN) was detective, as well as the proportion of CD4+/CD8+T cells, Th1/Th2and Tc1/Tc2; Oral administration of the LNT (800mg/kg) for10days, we observed the DTH mouse earswelling and the different changs of T-cell subsets.3. Effects of LNT on the T cell subsets of immunosuppressive miceBy given the LNT800mg/kg to CY suppressed immune function mice model for7days, observe the impacts of LNT on the αβ and γδ T cells subsets in SP, PPs, and MLN by flow cytometry.Resuls:1.75ng/ml PMA and Ionomycin to stimulate lymphocytes after5h, it shows that IFN-y and IL-4expression was significantly higher than the other two concentration groups; The efficacy of the protein secretion inhibition by BFA/Monensin Mixture (BM) is stronger than Monensin, so the expression of extracellular CD69was less than10%and intracellular CD69was more than90%. Here we suggest that BM, but not Monensin, is able to completely block the extracellular CD69on lymphocytes after in vitro stimulation with PMA/ionomycin.2. DNFB induced DTH mice, all the four tissues of the CD69+T cells was significantly increased; CD4/CD8ratio decreased significantly in SP and RLN, while the CD4/CD8ratio in PPs were increased significantly; and MLN showed no significant difference in each of the indexes. Thus, the Th and Tc cell subsets were not fully synchronized trends in the four parts of DTH mice, and Tcl (CD8+IFN-γ+) cells were characterized enhanced significantly.3Given with LNT for10days can reduce the DTH mice ear swelling; the CD4/CD8ratio in SP and PP decreased significantly, the MLN changed as the CD4/CD8ratio increased significantly. Th and Tc cell subsets changed different, IL-4reduced in SP while increased in RLN and MLN.4. Intraperitoneal injection of cyclophosphamide (CY),100mg/kg,24h after observed CY make αβ T cell proportion increased in PPs,MLN and SP. After fed with LNT, the PPs and MLN αβ T cell significantly higher than the CY group, while the proportion of γδ T cells in PPs return to normal level.Conclusion:1. Through the optimization of the detection of intracellular cytokines,75ng/ml PMA+1μg/ml Ionomycin were used to stimulate lymphocytes for5h in the presence of BFA/Monensin Mixture protein transporter inhibitor.2. Either the DTH mice model or the model of immunosuppression, the roles of LNT were different to the differeny parts of T cell subsets, especially to the T cell subsets in PPs and MLN. Moreover, LNT can modulate and recover the immune disfunction. Here we suggest that LNT may be related to the T cell redistribution in various tissues and related closely to the mucosal immune system.