| Fasciola gigantica(F.gantica)regulates immunity by continuously releasing the excretory-secretory products(ESP)in contact with the host immune microenvironment.Dendritic cells(DCs)are important antigen presenting cell(APC)in the host and play an important role in the immune process such as pathogen recognition and effector cell differentiation.The DC1,DC2 and DCreg subpopulations can induce helper T(Th)cells differentiate into Th1,Th2 and Treg cells,respectively.Therefore,the study of the induction of DCs differentiation by FgESP is helpful for us to understand how the Fasciola gigantica can regulate the immune response to the host of its excretory-secretory products.The aim at this study was to investigate the differentiation direction of monocyte-derived DCs(mo DCs)induced by FgESP in vitro and the possible relationship between the state of DCs and T cell differentiation.1.The direct effect of FgESP on the differentiation of DCs was determined.Different concentrations of FgESP were incubated with buffalo mo DCs in vitro for 48 h.RT-q PCR was used to detect the changes of surface molecular markers and cytokine gene expression in three subtypes of DCs.The results showed that compared with the PBS control group,the levels of DC1 marker CD86 and DCreg marker C1 QA and STAB1 m RNA were significantly down-regulated after stimulation with 200 μg/m L FgESP(P < 0.05);The expression of Th1 cytokine IFN-γ was significantly down-regulated,while the expression of Th2 cytokine IL-4 and IL-13 was significantly increased(P < 0.05).There was a doseeffect relationship between m RNA expression and FgESP concentration.It is speculated that FgESP can induce the differentiation of mo DCs to into DC2-type DCs in vitro.2.The effect of FgESP on the differentiation of DCs was indirectly determined by mixed lymphocyte response(MLR).Firstly,WST-1 method was used to determine the optimal cell number ratio of mo DCs co-cultured with lymphocytes at 1:5,Then,the ability of molybs stimulated by FgESP to stimulate lymphocytes was examined.the mo DCs pretreated with 200 μg/m L FgESP for 48 h were co-cultured with lymphocytes for 4 days in vitro.The mixed cells and culture supernatant were collected to extract total RNA from cells.RT-q PCR was used to detect Th cells phenotype-related cells.It was found that the m RNA levels of GATA3,FOXP3,IL-4,IL-10,IL-13 and TGF-β were significantly increased(P < 0.05).The main cytokines in the supernatant were detected by ELISA.It was found that the levels of IL-4,IL-10,IL-13 and TGF-β in FgESPpretreated group was significantly higher than those in control group(P < 0.05),which indicated that FgESP can promote lymphocytes to differentiate into Th2/Treg cells in vitro.3.In order to detect whether FgESP can induce DCs apoptosis,mo DCs were stimulated with 200 μg/m L FgESP for 48 h in vitro.The morphological characteristics and activity of DCs were evaluated by light microscopy,transmission electron microscopy and Hoechst33342 fluorescence staining.The results showed that stimulation of FgESP reduced the volume of mo DCs typical apoptotic characteristics such as pyknosis and nuclear fragmentation were found Chromatin.Caspase-3/7 kit was used to detect the activity of apoptosis-related enzymes.It was found that the intracellular caspase activity of mo DCs stimulated by FgESP was significantly higher than that in control group(P < 0.01),and positively correlated with the concentration of FgESP.RT-q PCR was used to detect the expression of apoptosis-related genes.It was found that the expression of anti-apoptosis genes Mcl-1 and Bcl-2 in mo DCs stimulated by FgESP was significantly lower than that in the control group(P < 0.01),while the expression of apoptosis-promoting gene Bax was significantly increased(P < 0.01).These results showed that FgESP can induce apoptosis of some DCs in vitro and has a concentration dependence of FgESP.In conclusion,this paper argues that FgESP can induce buffalo DCs to differentiate into DC2 in vitro,possibly by inducing apoptosis of some DCs,inducing the production of tolerant DCs,and promoting the differentiation of Lymphocyte into Th2/Treg cells.Combined with the previous research conclusions of the research group,this may be one of the mechanisms by which Fasciola gigantica regulates and escapes the host immune system by releasing FgESP. |
