The Distribution Of CKs And Expression Of Th9 Cell Of Early Echinococcus Granulosus Infection

Objective In vitro, the protoscolexes were co-cultured with mice spleen cells, and in vivo, a murine model of Echinococcus granulosus(E.granulosus) infection was established to verify whether the protoscolex induced the differentiation of Th1 cells, Th2 cells, Treg cells and Th9 cells from the spleen mononuclear cells. The expression of inflammatory cytokines was determined to check whether these Th cell subsets were involved in the mechanism of immune escape adopted by Echinococcus granulosus.Methods 1.In vitro experiments Spleen cells were isolated from BALB/c mice, and the protoscolex were co-cultured with mouse spleen cells for 12 h, 24 h, 36 h, 48 h. Flow cytometry was used to detect the number of spleen Th1 cells,Th2 cells, Treg cells and Th9 cells. The expression of IL-9, IL-10, IL-4, IFN-γ, TGF-β and IL-25 in culture supernatants was detected by ELISA. And the m RNA expression of IL-9Rα, RORα, IL-4Rα, IL-13Rα2, IL-5Rα of spleen cells were measured by the Real-time PCR. 2.In vivo experiments BALB/c mice were divided randomly into two groups. One group was infected with protoscolex to build the murine model of E.granulosus infection, and the other was given PBS with the same volume as control group. Mice were euthanized at Day 1, Day 5 and Day 9 after infection. Serum was collected and spleen cells were isolated as cell suspension. The number of Th1 cells,Th2 cells, Th9 cells and Treg cells in spleen cells was counted by Flow Cytometry. And the m RNA expression of IL-9, IL-10, IL-4, IFN-γ, TGF-β, PU.1 of spleen cells were measured by the Real-time PCR. Then, the expression of IL-9, IL-10, IL-4, IFN-γ, TGF-β, IL-25 in serum were determined by the ELISA.Results 1. In vitro experiments Compared with the Th subsets of the control group, the number of Th1 cells of the infection group had no difference at any post-infection time points, but the number of the Th2 cells and Treg cells were increased dramatically at 24 h-48 h, and the Th9 cells numbers were increased markedly at 36 h-48 h. The difference was statistically significant(P<0.05). Compared to the cytokine expression of the control group, IL-9 expression in culture supernatants was increased at 12 h, 24 h, 48 h(P<0.05), the expression of IL-10, IL-4, TGF-β was increased after 12 h-48 h(P<0.05), the level of IFN-γ was increased at 12-24 h, but was decreased at 36 h-48 h(P<0.05). IL-25 expression at 24 h only have a statistically significance(P<0.05). IL-9 was added into co-culture system at 48 h, the level of IL-9Rα, RORα, IL-4Rα, IL-13Rα-2 m RNA was increased with the increased infection time(P<0.05). 2. In vivo experiments A murine model of E.granulosus infection was successfully established. The number of Th1 cells were increased at Day 9 post-infection(P<0.05). And there was an increasing trend for the Th9 cells, Th2 cells and Treg cells ratio at Day 5 and Day 9 compared with those in the control group(P<0.05). The level of serum IL-10, PU.1, TGF-β, IL-25 was significantly higher that of the control group at Day 1, Day 5 and Day 9(P<0.05), the expression of IL-9 and IFN-γ was increased at Day 5 and Day 9(P<0.05). Cytokines protein expression level was consistent with the m RNA level. The level of PU.1 m RNA was showed increasing trend with the infection time(P<0.05).Conclusion The protoscolex stimulates the mouse spleen cells differentiate to Th1, Th2 cells, Treg cells and Th9 cells and up-regulates the secretion of cytokines, Different cell types and cytokines may interact to influence the host immune response. The results suggest that these Th cell subsets play a role in the immune escape of the hydatid disease.