| Objective:Malaria is a global public health problem with serious social harm and complicated pathogenesis.The main reason why malaria becomes a refractory disease is that the host lacks a long-term effective anti malarial immune response.The natural resistance to malaria depends on the repeated infection of Plasmodium,but the immune memory is difficult to maintain for a long time in malaria endemic areas.Because the malaria vaccine has not obtained the ideal immune protection,antimalarial drugs are still the main means to treat and prevent malaria.At present,Artesunate is the first-line drug for the treatment of severe malaria.Previous studies focused on dosage,duration of administration and observation of side effects of AS,but lack of studies on the immune protection mechanism of AS cured individuals in Plasmodium reinfection.At the same time,there are differences in the effect of AS in the experimental animal model.The way and time of AS administration may affect the therapeutic effect.Therefore,in this study,we screened the dose and time of AS administration,and applied the appropriate concentration of parasite infection,in order to obtain the AS cured mice of the susceptible mice(BALB/C)of Plasmodium yoelii 17XL(P.y 17XL).The resistance mechanism of AS cured mice in reinfection of P.y 17XL was further discussed in order to provide reference for the improvement of malaria adjuvant treatment and vaccine design.Methods:1.Animal model preparation:female BALB/c mice(8-10 weeks old)were randomly divided into Control group and Experimental group.Lethal Plasmodium yoelii 17XL was a new parasite infected red blood cells collected from mice inoculated with the first generation of parasite protected by liquid nitrogen.The mice in the Experimental group were infected by P.y 17XL(1×10~4/0.1mL,i.v)and given AS orally(25mg/kg/d)for 6 days when parasitemiawas less than 10%.On the day 30after infection,the surviving mice were infected with P.y 17XL(1×10~6/0.1mL,i.p.)for the second time.At the same time,the Control group(P.y 17XL susceptible mice)and the Experimental group(P.y 17XL resistant mice)was infected by P.y 17XL(1×10~6/0.1mL,i.p.)on the day 60 after infection.2.The thin blood smear of tail vein was prepared from the d1 of infection and the parasitemia,survival rate,body weight and clinical score were observed dynamically.3.Paraffin sections were prepared from spleen and liver tissues on d5 of infection,and histopathological changes of spleen and liver were detected by HE staining.The infiltration of inflammatory cells was observed by F4/80 Immunohistochemical staining.4.Splenic lymphocyte suspension was prepared on d5,the number of CD4~+and CD8~+T cell subsets,the expression level of CD44,CD62L and CD127,the expression level of IFN-γand TNF-αsecreted by CD4~+and CD8~+T cells,and the expression level of GB secreted by CD8~+T cells were detected by flow cytometry.5.Hepatic lymphocyte suspension was prepared on d5,Flow cytometry was used to detect the number of CD4~+T cell and CD8~+T cell and the expression level ofCD127,the expression level of IFN-γsecreted by CD4~+T cell and CD8~+T cell and the expression level of GB from CD8~+T cells.6.The serum IgM and IgG antibody levels were detected by ELISA.Results:1.In the Experimental group,mice infected with P.y 17XL were treated with AS,the parasitemia was controlled at about 6%.When the drug was stopped,pRBC could still be seen under the microscope,parasitemia could not be detected on d15,and the survival rate of mice was 90%.These results suggest that AS can effectively control the proliferation of Plasmodium.In order to observe whether the cured rats of AS have resistance to P.y 17XL infection,the surviving rats received the second attack of P.y 17XL(d30).pRBC was detected in the peripheral blood of mice infected on d5,the infection rate of RBC was less than 5%,parasitemia could not be detected on d10,and the survival rate of mice reached 70%.In conclusion,AS cured mice obtained immune protection against reinfection of Plasmodium.2.In order to verify the resistance of AS cured mice to P.y 17XL infection,the Experimental mice received the third infection(d60)of P.y 17XL,while the Control mice without AS treatment received the same dose of P.y 17XL infection.From the infection of d3,the Control group mice showed decreased activity,fur fold,pale toes.The clinical score was significantly higher than that of the Experimental group.The results of dynamic monitoring of parasitemia showed that the parasitemia in the Control group increased rapidly from d3 to the peak of 87.5%on d8.When the infection rate of red blood cells in the Control group reached the peak,the weight of the mice decreased significantly,the urine was red or dark green,the body arched,the response to stimulation was slow,the mice begain to death from d6 and all the mice died on d8.On the contrary,the mice in the Experimental group were normal,no pRBC can be seen under the microscope,and all the mice survived in the observation period.The results showed that the Control group was susceptible to P.y 17XL,while the Experimental group was resistant to P.y 17XL.It is suggested that Artesunate is helpful for the establishment and maintenance of host immune memory to non severe malaria infection.3.In order to find out the effect P.y 17XL infection on spleen and liver,we made pathological observation on the two kinds of tissues.The weight of spleen and liver in the Control group increased significantly(P<0.0001,P<0.05),and the tissue swelling was obvious.Histopathological analysis showed that the spleen structure of the Control group was disordered,the spleen nodules disappeared,the red and white pulp boundary disappeared,the malarial pigment deposited and the tissue structure was seriously damaged.In the Experimental group,the structure of spleen was clear,the boundary between red and white pulp was clear,and there was malaria pigment deposition in the red pulp.In the Control group,the hepatocytes were swollen extensively,there were malaria pigment deposition and a large number of inflammatory cells infiltration in the central vein.In the Experimental group,the structure of liver was clear,hepatocytes were closely arranged,and a small amount of malarial pigment particles can be seen.The results of F4/80 immunohistochemistry showed that there were a lot of macrophages infiltrated in the Control group,while the inflammatory reaction in the Experimental group was lighter.The results showed that the spleen and liver were affected by P.y 17XL infection,and the pathological damage of liver and spleen tissue in mice cured by AS was significantly relieved.This is closely related to the effective control of RBC infection rate in the Experimental group.4.The contents of CD4~+and CD8~+T cells in spleen and liver were analyzed.Compared with the Control group,the percentage of CD4~+T cells and CD8~+T cells in the spleen of the Experimental group increased significantly(P<0.05,P<0.01),and the percentage CD4~+and CD8~+T cells in the liver also increased significantly(P<0.05).Furthermore,the contents of spleen effector T cells(Teff,CD44~+CD62L~-)and central memory T cells(Tcm,CD44~+CD62L~-)were detected.Compared with the Control group,the content of Teff and Tcm in CD4~+T cells in the Experimental group increased significantly(P<0.001,P<0.05).The content of Teff and Tcm in CD8~+T cells was also significantly higher than that in the Control group(P<0.05,P<0.01).The results showed that CD4~+and CD8~+T cells in the spleen and liver of Artesunate treated mice were involved in the protective immune response to malaria.5.IFN-γand TNF-αare important effectors of cellular immune response andplay an important role in antimalarial protection.Therefore,we analyzed the expression of IFN-γand TNF-αin spleen T cells.Compared with the Control group,the content of CD3~+CD4~+IFN-γ~+T and CD3~+CD8~+IFN-γ~+T cells in the Experimental group were significantly increased(P<0.05,P<0.01).In the Experimental group,IFN-γ~+cells accounted for 51.2%of CD3~+CD8~+T cells,and IFN-γ~+cells accounted for8.9%of CD3~+CD4~+T cells,it is suggested that CD8~+T cells in the spleen are important effector cells for IFN-γsecretion.Furthermore,we analyzed the level of IFN-γ~+TNF-α~+T cells.Compared with the Control group,the content of CD3~+CD4~+IFN-γ~+TNF-α~+T and CD3~+CD8~+IFN-γ~+TNF-α~+T cells in the Experimental group were significantly higher(P<0.01).In the Experimental group,IFN-γ~+TNF-α~+cells accounted for 24.5%in CD3~+CD8~+T cells,and IFN-γ~+TNF-α~+cells accounted for5.7%CD3~+CD4~+T cells.The results showed that AS induced antimalarial immune protection was mediated by Th1 and Tc1(Th1 like CTL)subsets by secreting IFN-γand TNF-α.6.In order to analyze the difference of effect or effect memory T cell subsets inIFN-γproduction,the CD127 expression level of IFN-γ~+T cells was analyzed.Compared with the IFN-γ~+effective memory T cells in Control group,the contents of CD4~+IFN-γ~+CD127~+T cells and CD8~+IFN-γ~+CD127~+T cells in spleen and liver increased significantly(P<0.05).It is suggested that effector memory T cells are an important source of IFN-γcells.Further analysis of IFN-γ~+effector T cells showed that the proportion of CD4~+IFN-γ~+CD127~+effector T cells in the spleen and liver of the Experimental group was not significantly different from that of the Control group,but the content of CD8~+IFN-γ~+CD127~+effector T cells increased significantly(P<0.01).The total number of IFN-γ~+cells in the Experimental group was analyzed,the total number of CD8~+T cells secreting IFN-γwas significantly higher in spleen(P<0.05)and liver(P<0.01).It further strengthened the role of CD8~+T cell immunoregulation in the anti malarial immune response.7.Granzyme B is a cytotoxic factor of CTL cells.Therefore,we analyzed the level of GB secreted by CD3~+CD8~+T cells in spleen and liver.Compared with the Control group,the CD3~+CD8~+T cells in the spleen of the Experimental group showed almost no expression of GB,and the percentage and absolute number of CD3~+CD8~+GB~+T cells decreased significantly(P<0.001,P<0.05).On the contrary,the CD3~+CD8~+T cells in the Experimental group and the Control group could express GB,and there was no significant difference in the number of CD3~+CD8~+GB~+T cells between the two groups.The results showed that the cytotoxic effect molecule GB of Artesunate was mainly derived from CTL cells of liver rather than spleen.This further strengthens the important role of liver in clearing pRBC.8.Humoral immune response is an important role of cellular immune response.Therefore,we detected the level of Plasmodium specific antibody in the serum of the two groups of mice.According to the results of standards,the IgM standard curve log log(y)=0.162×log(x)-0.341(R~2=0.951)and IgG standard curve log(y)=0.326×log(x)-0.088(R~2=0.944)were drawn.There was no significant difference in serum IgM level between the Control group and the Experimental group,but the level of IgM in the Experimental group(3rd)was significantly higher than that in the Experimental group(2nd)(P<0.05).Compared with the Control group,the serum IgG level of the 2nd and 3rd of AS cured rats were significantly higher(P<0.001),and the3rd were more significantly higher than the 2nd(P<0.05).The results showed that the level of antibody response to P.y 17XL infection was significantly improved in AS treated mice.Conclusion:1.Low density parasite infection combined with Artesunate treatment is helpful for BALB/c mice to obtain immune protection against reinfection of P.y 17XL.2.Cellular immunity mediated by CD4~+T cell and CD8~+T cell plays an important role in Artesunate treatment of mice against reinfection of P.y 17XL.3.IFN-γand TNF-αfrom spleen and liver T cells,GB was secreted by CTL and serum specific IgG antibody are important cytokines against P.y 17XL infection. |
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