Study On The Function Of Macrophages And Dendritic Cells Activated By Schistosoma Japonicum High Mobility Group

Schistosomiasis is most commonly found in Africa, as well as Asia and South America. The disease is a parasitic disease sond only to malaria. A long-term control of schistosomiasis was absolutely depend on the application of high effective and safe vaccines against schistosoma. Radiation-attenuated cercariae or schistosomula vaccine(RAV) is recognized as one kind of vaccine with high protection effect,nevertheless it can’t be widespreadly applied to the schistosomiasis control due to the difficulty of material source and safety problems. Therefore, in order to develop molecular vaccines with high efficiency and safety against schistosome, it is needed to study on the high protective effect mechanism of RAV model.Many studies found that higher anti-infective effect could be induced in variety of animal models vaccinated with RAV and the dominant Th1 response was appeared in host. The reason may be the fact that radiation-attenuated schistosomula(RAS) is damaged seriously after irradiation, the migration of RAS is retarded in the lung of hosts and large amounts of molecules were released from schistosomula to the hosts.Eventually, those situations induced the stronger immune responses in the hosts. High mobility group protein B1(HMGB1) is a kind of nuclear protein, it can move from nucleus to extracellular microenvironment under the condition of the ionizing radiation. Previous studies have shown that schistosoma mansoni HMGB1 can induce release of large amounts of inflammatory cytokines from macrophages of host. It is one of important molecules that elicit proinflammatory response in hosts So we speculated that schistosoma japonicum HMGB1(SjHMGB1) from schistosomula plays a similar important role in the high protection of RAV. This study aims to explore the functions of SjHMGB1 in activation of immune cells and regulating immune responses in mice, and laid a foundation for further clarifying immune functions of SjHMGB1 in RAV.1 、 We analyzed the expression of SjHMGB1 in RA and normal schistosomula-derived cells. The schistosomula-derived cells were acquired by digesting the RA and normal schistosomula with trypsin in different stage of schistosomula. The results revealed that, the expression level of SjHMGB1 was significantly increased in RA schistosomula-derived cells at 4d by flow cytometry(FCM), compared with normal schistosomula-derived cells, and significantly decreased at 7d(P<0.01). But at 10 d and 14 d, there was no significant difference. The expression of SjHMGB1 at the mRNA level in different stages of RA and normal schistosomula was tested by qPCR, we found that SjHMGB1 mRNA expression in RA schistosomula at 4d and 7d was significantly increased compared with normal schistosomula, and at 10 d there was no significant difference between two groups. So we preliminary inference that SjHMGB1 was higher expressed and released in the early RA schistosomula.2 、 We analyzed the reaction of sensitized lymphocyte induced with reSjHMGB1.The sensitized lymphocyte had been stimulated with reSjHMGB1 protein for 4h.MTT method was used to assay the proliferation of sensitized lymphocyte. The subgroup of sensitized lymphocyte and the cytokines sreted by sensitized lymphocyte were detected by FCM. The results showed that reSjHMGB1 could stimulate the sensitized lymphocyte’s proliferation(SI>2);The value of ratio of CD4+/CD8+T from group immunized with reSjHMGB1 protein was 2.28,significantly higher than that of control group(P<0.01) which was 1.76. the expression of IFN-? was also increased significantly(P<0.01).These results suggest that SjHMGB1 could elicit proliferation of sensitized lymphocyte and enhance polarization of CD4+ T cells to Th1 phenotype.3 、 We analyzed the immune regulation function of reSjHMGB1 in innate immunity.RAW264.7 cells were incubated with reSjHMGB1 protein for 48 h.The expression of surface molecules on macrophage RAW264.7 was detected by FCM and extracellular cytokines were detected by ELISA. The results showed that SjHMGB1 could significantly up-regulate the expression of Toll-like receptor 4(TLR4)on RAW264.7 cells(P<0.01), but the expression of Toll-like receptor 2(TLR2) was significantly down-regulated(P<0.05), the sretion of TNF-α, IL-1β and NO from RAW264.7 cells stimulated with reSjHMGB1 was also increased significantly(P<0.01). These results suggested SjHMGB1 could induce differentiation of macrophage(RAW264.7) to the proinflammatory M1 type via activation of TLR4 signal pathway.4、We studied that reSjHMGB1 induce functional maturation of mouse dendritic cells(DC). mDCs were stimulated with reSjHMGB1 for 48 h, then the cultured cells were incubated with CD4+T cells for 168 h. Expression level of chemokine receptors and intracellular cytokines as well as the proliferation of T cell were detected by FCM,and extracellular cytokines were detected by ELISA. The results showed that DCs stimulated with reSjHMGB1 could significantly increase the expression of chemokine receptors CCR7 and CXCR4(P<0.01), as well as the intracellular cytokines TNF-α,IFN- ? and IL-4(P<0.01). reSjHMGB1 could enhance CD4+T activated by DC stimulated with reSjHMGB1 protein to release large amount of IFN-?(P<0.01). These results suggested that SjHMGB1 could promote the functional maturation of DC,activate CD4+T cell and induce Th1 immune response.5、We preliminarily explore the activation of neutrophil induced by reSjHMGB1.We found the formation of Neutrophil extracellular traps(NETs)after reSjHMGB1 protein had stimulated neutrophil for 4h by Fluorescence microscopy. We detected the mRNA level of cytokines by qPCR, and found that MMP-9, MCP-1,CCL-3 and CXCL-2 mRNA levels of group stimulated with reSjHMGB1 protein were significantly increased than that of control group(P<0.01). The results suggested that SjHMGB1 could activate neutrophils to the inflammatory phenotype.In conclusion, in this study we found that SjHMGB1 was highly expressed inearly RA schistosomula. reSjHMGB1 protein could induce the maturation of macrophage and DC, as well as activation of CD4+T cell, and enhance Th1-biased response of immune cells.We also explored the forming process of NETs stimulated by reSjHMGB1.This study will provide a basis for further clarifying the immunobiological functions and mechanisms of SjHMGB1 in RAV model.