The Interaction Of Plasmodium Yoelii Sporozoites And Bone Marrow-dendritic Cells

Malaria, one of the human infectious diseases with high incidence and fatality rate, is transmitted through the bite of infected Anopheles mosquito. Vaccine is one of the effective ways to control the morbidity and the prevalence. Although the attenuated sporozoites vaccine can induce effective protection, it cannot be extensively used in epidemic area ,because it's hard to produce. Thus, preparation of the effective subunit vaccine is the present focal point, and thoroughly investigation of immunologic mechanism of preerythrocytic stage is the prerequisite to design effective preerythrocytic stage subunit vaccine.Dendritic cell(DC) ,the professional antigen presentation cell(APC), is a natural bridge connecting the innate immunity and the adapted immunity to Plasmodium. Recent studies show that sporozoites enter the skin after the bite of Anopheles, some sporozoites trip to lymph vessels. In addition, DCs in the skin-draining lymph nodes were essential for activation of preerythrocytic stage sepcific CD8+T cells, which was the key immune effector to clear pre-erythrocytic stage in hepatocyte. Thus, sporozoites travel to the lymph vessels might be important in inducing the DC maturation. However, it is still unknown how the sporozoites induce the DC maturation.In our study, we firstly constructed a platform---co-culture of BMDCs and sporozoites in vitro---to observe whether sporozoites can initiative invade the DC. Secondly, a fluorescence microscope was used to observe the interaction of the DCs and sporozoites. Then the costimulatory molecules(CD80,CD86) and MHC-â…¡expressed on DCs were detected by flow cytometry to detect whether sporozoites can induce DC maturation. Finally, recombinant plasmid pFLAG-CMV8-CSP was transfected into DCs and investigate its role on DC maturation. The contents and results include the following two aspects: 1. Sporozoites can induce DC maturation.1) Morphology of DCs and sporozoites after co-culture: DCs were induced by GM-CSF from mouse bone marrow in vitro, and co-cultured with sporozoites dissected from Plasmodium yoelii EGFP-BY265 infected Anopheles stephensi. 2h, 4h and 24h later, the interaction of the DCs and sporozoites were observed under fluorescence microscope. The results showed that most of the sporozoites can initiative invade DCs and transform into spherical early erythrocytic stage like forms at 24h, indicated that sporozoites can develop in DCs.2)The invaded sporozoites induce DCs maturation: DCs were induced by GM-CSF from mouse bone marrow in vitro, and co-cultured with sporozoites dissected from Plasmodium yoelii BY265 infected Anopheles stephensi. 2h, 4h and 24h later, the costimulatory molecules(CD80,CD86) and MHC-â…¡expressed on DCs were detected by flow cytometry. Although the expression of costimulatory molecules on DCs was not affected at 2h, sporozoites significantly up-regulated the expression of costimulatory molecules on DCs after 4h and 24h incubation with DCs. The result shows that invaded sporozoites can induce DCs maturation, which provided a crucial prerequisite to induce the activation of CD8+T cell.2. Transfected recombinant plasmid pFLAG-CMV8-CSP induces DC maturation.Circumsporozoite protein(CSP) is one of the crucial surface protein on sporozoite. Naturally, sporozoites can invade KC mediated by CSP and TRAP(thrombospondin-related adhesive protein), which hide itself in parasite vacuole to avoid killing by phagolysosome. Interestingly, sporozoite can uses a PEXEL motif to introduce the CSP into the hepatocyte cytoplasm. We investigated the role of CSP in the DC maturation induced by sporozoites.The recombinant plasmid pFLAG-CMV8-CSP was constructed and transfected into DCs, and the costimulatory molecules(CD80,CD86) and MHC-â…¡expressed on DCs were detected by flow cytometry 24h later. The results showed that the transfection efficiency amount to more than 40%. Although the expression of the MHC-â…¡not affected, the costimulatory molecule, such as CD80, were significantly up-regulated. This data indicate that CSP in intracytoplam can induce DC maturation, but may not through the pathway mediated by the MHC-â…¡.Conclusively, we are succeed in constructing the platform to investigate the interaction of bone marrow-dendritic cells and Plasmodium sporozoites in vitro. In addition, we found most sporozoites could invade the DC and induce its maturation, and CSP might play an important role in DC maturation induced by sporozoites. The result will help us understand the interaction of bone marrow-dendritic cells and Plasmodium sporozoites, and establish an important foundation to investigate the contribution of DCs in preerythrocytic stage specific innate immune response.