| Malaria is transmitted by anopheles mosquitoes and caused by the infection of different Plasmodium species,including plasmodium falciparum,Plasmodium malariae,Plasmodium ovale,Plasmodium vivax and plasmodium knowlesi.Among many infectious disease,malaria cause high morbidity and mortality rate in worldwide.According to the world malaria report,216 million cases of malaria in total were reported from 91 countries,and the global death toll from malaria was estimated as 731,000 in 2016,roughly the same number as in 2015.After the mosquito bite,the parasite sporozoite are injected into the host.Then sporozoites enter the blood circulation and can quickly invade the liver cells within minutes.A successful sporozoite can produce 10,000-30,000 offspring sporozoites within the hepatocytes in 5-8 days.Following the rupture of merozoites in the liver,sporozoites began to invade RBCs and replicate the asexsul stage about 48 hours in the blood circulation for Plasmodium vivax,Plasmodium ovale and Plasmodium falciparum,while Plasmodium malariae takes about 3 days and Plasmodium knowlesi need only about 1 days.Malaria clinical symptoms are usually characterized as periodic onset of cold fever,the main complications of the disease include CM,severe anemia,acidosis,hypoglycemia,HMS and toxic shock.In recent years,the many malaria researches have focused on the adaptive immune response of malaria infection which are critical for the vaccine development.It's clear that the processes and mechanisms of immune response cuases by malaria are complicated and involved many differents immune cells.Studies have showed that prior to the adaptive immune response,the main immune response for malaria infection relies on natural immune cells,such as NK cells,NKT cells,DC cells and ??T cells.These cells are crucial components of innate immune cells to supporting the body against the malaria parasite during in the erythrocytic stage.Importantly,the natural immune cells surface located TLRS(toll-likereceptors)receptors are important pattern recognition molecules,and mediated signals transduction for the natural immunity response.Previous studies have demonstrated that TLRs expressed in a variety of innate immune cells including macrophages,DC cells,B cells and ??T cells,and these innate immune cells could recongnize the invading pathogenic microorganisms by TLRs.TLRs are essential in identification of pathogen-associated molecular patterns(PAMPs)and infectious agents in activating innate immune responses.The TLRs ligand is a natural macromolecular component derived from pathogens,consisting of lipids,proteins and nucleic acids.Many TLRs have been identified,for example,TLR3?TLR7?TLR8 and TLR9 are located in the endosome and recongnize nucleic acids such as viral DNA or RNA.In response to the malaria infection,TLR2 TLR4 and TLR9 have been proved to be involved in the process of host natural immunological identification of erythrocytic stage.To date,however,it is not clear whether other TLRs are involved in the immune response of Plasmodium yoelii,particularly the TLRs in gamma delta T cells.To explore the protective immune response of malaria infection,we characterized in details of gamma delta T cells in the spleen during the infection of Plasmodium yoelii NSM of C57BL/6 mice.In this paper,we observed the siginificant changes of ??T cells in the spleen after the infection of Plasmodium yoelii NSM in mice.These changes include the percentage and absolute number of ??T cells,??T cell activation and the level of cytokines.In addition,the Plasmodium yoelii NSM infected ??T cell knock out mice provided the further supporting evidences of the ??T cell in the pretection of plasmodium infection in C57BL/6 mice.The main results in this study include.(1)Successful establishment of Plasmodium yoelii NSM infected the C57BL/6 mice model: The i RBC was injected into C57BL/6 female mice aged 6-8 weeks by tail intravenous injection.After 24 hours of infection,the blood from the tip of the tail was collected for blood slide preparation.The parasitemia was checked by microscope.After D2 of infection,low level of i RBC were observed.At D3,the level of parasitemia in the infected group reached to 6.495 ± 2.709%.The level of parasitemia was peaked first at D5(14.1425 ± 4.333%)and the parasitemia gradually increased and reach the maximum at day 18(63.875 ± 20.694%,figure 1B).Further analysis of the HE staining of normal and infected spleen showed significant pathological changes in infected tissue,indicating an important role of spleen in control and the establishment of chronic Plasmodium yoelii infection.(2)Alteration of CD3+??TCR+ cells in Plasmodium yoelii infeced spleen: The mesenteric lymph nodes,lung,liver,spleen,peripheral blood lymphocyte were collected from C57BL/6 mice(infected & control),and the single cell suspension was prepared respectively.CD3+??TCR+cells in different organs were counted by flow cytometry using CD3 and ??TCR monoclonal antibody.The results showed that the increase of CD3+??TCR+ cells in the spleen was significantly higher than the control group,indicating that the CD3+??TCR+ cells in the spleen play a crucial role during Plasmodium yoelii infection.(3)Detailed anaylsis of CD3+??TCR+ at different days of infection: The spleen cells were collected from Plasmodium yoelii NSM infected C57BL/6 mice as well as normal control mice at D2,D4,D6,D8,D10 and D14.The percentage and the absolute number of CD3+??TCR+ cells were detected by cell surface staining.Our study showed that it was at D14,the CD3+??TCR+ cells had a significant change in infected mice.(4)Intracellular cytokine profiles in infected spleens: In order to deeply understand the ability of CD3+??TCR+ cells in secreting a variety of cytokines in spleens,we performed the in vitro experiment of the spleens cells co-cultured with stimulants PMA.The intracellular cytokine level of IL-4,IFN-?,IL-17,IL-22,IL-10 and IL-5 was measured by FACS.In this study,we showed that the IL-4 + cells on CD3+ ??TCR+ cells were increased,while IL–10+ cells and IFN-?+ cells were reduced in infected spleen.There was no change for IL-5+ cell and IL-22+ cells.These results suggested that CD3+??TCR+ cells play a vital role in the process of infection,and the ??T cells can secrete less Th2 type cytokines(IL-4?IL-5)than Th1 cytokines(IFN-gamma,IL-10)and less TL17 cell cytokines(IL-17?IL-22)(Fig.6).Furthermore,the level of CD69 in CD3+??TCR+ cells were found to be increased after infection,while the expression of CD40 L and CD62 L were decreased,and CD25+ in CD3+??TCR+ cells have no obvious changes.It indicates that CD3+??TCR+ were significantly activated in the spleen after infection.The expression of CD4 and CD8 on the surface of CD3+??TCR+ cells were extremely lower and there were no significant differences between the normal mice and infected mice.It is further validated that CD3+??TCR+ cells usually can not express CD4 and CD8.It has been reported that the CD3+??TCR+ cells can regular express the V?2 and ICOS and they were notably expressed lower after infection.In addition,CD3+??TCR+ cells can also express PD1,PDL1 and PDL2.After infection,a significant rise in the proportion of PD1 on CD3+??TCR+ cells were observed.The above results show that CD3+??TCR+ cells maybe as the feedback mechanism of excessive inflammation during severe infection.Finally,the expression of CXCR3 and CX3CR1 were not changed on CD3+??TCR+ cells after infection(Fig.5).The reason of this phenomenon was not clear,the further studies are needed.(5)Further evidences from ??T knockout mice: The normal C57BL/6 mice and ??TCR knockout mice were infected with Plasmodium yoelii NSM by tail intravenous injection as before.The results showed that the parasitemia in wild type mice and ??T knockout mice rapidly increased in a few days and then gradually decreased after infection.However,the parasitemia in ??T knockout mice(3.180 ± 1.094%)was significantly higher than wild type mice after infection(*P<0.01,1.270 ± 0.5241%).It suggested that ??T cells were involved in the immune response in the early stage of infection.(6)TLR2,TLR3,TLR4 expression in CD3+??TCR+ cells: TLR2,TLR3,TLR4 expression in CD3+??TCR+ cells were further checked.The results showed that TLR2,TLR3,TLR4 expression are increased in CD3+??TCR+ cells,indicating the activation of the relevant receptors on spleen cells during plasmodium infection in mice.Further studies are required to understand the interaction between hosts and parasites through the regulating the expression of TLRs. |
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