| Objective: Malaria is a parasitic protozoan infectious disease which is caused directly by the infection of Plasmodium.In World Health Organization(WHO)malaria report for2019,the number of new cases of malaria dropped significantly in 2018,but there were still 228 million new cases,killing about 400,000 people,more than half of them were children under the age of five.The research and development of protective vaccines against malaria is particularly important,but so far there is no fully effective anti-malarial vaccine,and the world's first malaria vaccine approved by the European Drug Administration-RTS,S/AS01-is up to 50 per cent effective,so it is necessary to fully understand the characteristics of malaria immunization,which is of great significance to the research and development of malaria vaccine and to the effective prevention and control of malaria.In the malaria immune response,T lymphocytes play an important role in the anti-hepatic and blood stage infections of Plasmodium.Antigen stimulates T cells,which quickly become effector T cells,and eventually most of the T cells undergo apoptosis and are removed by the body.Plasmodium infection is found to have immunosuppressive phenomenon,because of the existence of inhibitory factors,Plasmodium infection will accelerate the terminal effect differentiation process of T cells,inhibit the persistence of T cell immune memory,specific T cell reduction.In recent years,the role of programmed cell death receptor-1(PD-1)and its ligands signaling pathway in malaria infection have attracted much attention and may play an important role.Studies have shown that multiple chronic infections and tumor development are closely related to T cell exhaustion.Current studies have shown that PD-1 is able to mediate functional depletion of T cells in malaria infection.Recent studies have also found that malaria parasite infection not only induces functional depletion of T cells but also causes depletion of B cells,suggesting that the presence of PD-1 may potentially block effective control of malaria infection.However,the role of the PD-1/PD-L1 pathway in malaria's initial immune response and the formation and maintenance of immune memory and its mechanisms are not clearly reported.Therefore,we used Plasmodium berghei ANKA(Pb ANKA)to infect BALB/c mice, used PD-1 monoclonal antibody to block the function of PD-1 to analyze the effect of PD-1 deletion on immune effector cell function,and explored the possible mechanism of PD-1 in malaria initial immune response and the establishment of immune memory,with a view to providing a new theoretical basis for developing effective vaccines to control malaria pathogenesis.Method: 1.Experimental animal and models of construction.Primary infection group:Female 6–8-weeks-old BALB/c mice were infections with 1×106 Pb ANKA parasitized erythrocytes(p RBC)by intraperitoneal(i.p.).Reinfection gruop: Female 6–8-weeks-old BALB/c mice were infections with 1×106 Pb ANKA parasitized erythrocytes by i.p.,constructed different experimental animal models.The drug treatment of chloroquine was carried out in 4-5 days after infection.30 days after infection,immunization of the same plasmodium was carried out.PD-1 monoclonal antibody blocked PD-1 function in primary infection group.Pb ANKA malaria infected mice began to use antibody of PD-1on 0 day,3day,5day and 7day(200?g i.p.)after primary infection.PD-1 monoclonal antibody blocked PD-1 function in reinfection gruop.Pb ANKA malaria infected mice began to use antibody of PD-1 on 0 day,3day,5day and 7day(200?g i.p.)after reinfection infection.The Control Group was injected with the same dose of PD-1monoclonal antibody at the same time.Each group consisted of nine mice.2.Count parasitemia level as well as survival rates.At different time points of Pb ANKA infection,the mice were made into thin blood membrane by the way of caudal vein blood collection.after Giemsa staining,the parasitemia level of each group of mice was counted,the level of parasitemia was dynamically monitored,the death of each group of mice was recorded,and the survival rate of each group of mice was calculated.3.Preparation of splenocyte suspension.On the 5th day after each group of mice were infected,the spleen was removed aseptically in the super-clean workbench,and the spleen was made into splenocyte suspension by grinding.Spleen cells were cultured using a 24-hole culture plate(FALCON)with 500?l of splenocyte suspension(final concentration of 1×107/ml)in each hole.Each sample was added with 3 holes.After 48 hours of culture,spleen cells were collected and centrifuged at 350 g for 10 min.Spleen cells were collected for culture supernatant and stored in a refrigerator at-80? for later detection of cytokines determination.4.Serum separation.On the 5th day after the initial infection and the 12th day after the reinfection,the whole blood of the mice in each group was preserved in 1.5ml centrifuge tube.The whole blood of the mice in each group was left at room temperature for 1-2 hours and overnight at 4°C,3000 rpm,10 min,the serum is separated into a new centrifuge tube,labeled,and stored in the refrigerator at-80? for subsequent antibody detection.5.Fluorescent antibody staining and flow cytometry analysis.Prepared splenocyte suspension were used to cell surface staining: negative control tubes,single dye tubes and laboratory tubes were used,Each tube was added with washed 1×106 splenocytes.The negative control tube was added with the same type of control antibody.the experimental tube was added with 20?l of the corresponding m Ab.After shaking and mixing,the cells were incubated for 15-20 minutes at room temperature to avoid light.2000 g centrifuged for 5 min,discarded the supernatant and added 0.5 ml PBS to be tested on the computer.Prepared splenocyte suspension were used to intracellular staining: negative control tubes,single dye tubes and laboratory tubes were used,PMA(50 ng/ml),Ionomycin(1?g/ml)and BFA were added in the tubes to stimulate for 4-6 hours.After 3 times of PBS washing,the isotype control antibody was added to the negative control tube,and the surface-labeled m Ab was added to the experimental tube.After 3 times of light-free washing,the PBS was incubated for 30 min.After 2 times,the 200?l broken film solution was added at 4? to avoid light to break the membrane for 30 min.Discard the supernatant and add PBS 500?l for on-board detection.Using flow cytometry(FACS Celesta,B&D),the excitation wavelength is488 nm,to obtain the cell using FACS CELLQUEST software,analysis of 100000 cells for each sample,negative control tubes were used to determine the voltage,and single dye tubes were used to adjust the compensation,using forward scattering angle(FSC)and lateral scattering angle(SSC)to determine the cell population,with negative control for reference,the nonspecific fluorescence shown to take care of 99% as the background deduction,to display two-dimensional bitmap,record the percentage.6.Detection of cytokines and antibodies.The levels of IFN-?,TNF-?,IL-10 and IL-6 in culture supernatantswere measured by commercial enzymelinked immunosorbent assay(ELISA)kits according to the manufacturer's protocol(R&D Systems,Minneapolis,MN).The OD values were read in a microplate reader at 450 nm.The concentration of all cytokines in samples were calculated against the standard curve generated using recombinant cytokine by using Soft Max Pro 4.3.1LS(pg/ml).Preparation of Pb ANKA whole worm antigen,expression and purification of recombinant MSP119 protein,detection of specific antibody level against Pb ANKA whole worm antigen and MSP-119,detection of OD value at 450 nm,use of automatic enzyme labeling instrument.7.Detection of splenocyte cytokine m RNA expression levels.The total RNA of spleen cells was extracted by Trizol method,the g DNA was removed from the RNA,and the m RNA expression levels of IL-10,IL-6 and IL-21 were detected by Real-time PCR.8.Statistical analysis.SPSS software was used to analyze all the data,the version used was 23.0(SPSS inc.,USA).Using the one-way ANOVA test to compare and analyze the mean differences across groups.All data were presented using the mean ± standard error(SEM)method.P <0.05 indicated that there was a statistical difference.Results 1.The effect of PD-1 on the primary infection of Pb ANKA.On the 6th day after infection,the red blood cells infected by Plasmodium began to appear in the peripheral blood of BALB/c mice infected with Pb ANKA,and then the level of parasitemia increased rapidly and continuously.The parasitemia reached 40% on the 17 th day after infection,while mice began to die on the 15 th day after infection.All mice died after23 th day after infection.However,BALB/c mice infected with Pb ANKA died on the13 th day after infection with monoclonal antibody against PD-1,and all mice died after18 th day after infection.On the 5th day after infection,the expression of splenic conventional dendritic cells(c DCs)and plasmacytoid dendritic cells(p DCs),MHC?,CD86,and PD-L1 were detected by flow cytometry on DCs.We found that c DCs and p DCs showed a significant increase in normal infection.After the use of PD-1 m Ab,the number of c DCs decreased significantly,the p DCs did not change significantly,and the expression levels of CD86 and MHC?on CD11c+DCs were not different in the m Ab blocking group and the normal infection group.But the level of PD-L1 expression on c DCs was significantly increased after monoclonal antibody blockade.Effects of PD-1blocking on the number of regulatory T cells(Tregs),bone marrow-derived inhibitory cells(MDSCs),Th1,activated T,follicular helper T cells(Tfh)in mouse splenocytes on day 5 after infection were detected by flow cytometry.Cytokines were detected by ELISA.Elimination of PD-1 had no obvious effect on the activated T and Th2 of Treg,Tfh,while the number of MDSCs increased significantly,and the level of Th1 decreased significantly in PD-1 blocked group.m RNA analysis of spleen cells showed that IL-21 and IL-6m RNA decreased significantly,but IL-10 did not change significantly.Then the cytokines in the supernatant of spleen cell culture were detected.It was found that TNF-?did not change significantly after PD-1 elimination,but IFN-?,IL-10 and IL-6 were significantly decreased.Effects of PD-1 blocking on the number of TCM,TEM,short-lived,long-lived plasma cells were detected by flow cytometry.Serum antibodies specific to Pb ANKA antigens Ig G1 and Ig G2 a and serum antibodies specific to recombinant MSP119 antibodies were detected by ELISA on day 5 after infection.For immune memory,we found that on the 5th day after infection,TCM and short-lived plasma cells decreased.On the 12 th day after infection,TEM and short-lived plasma cells did not change.Serum antibodies specific to Pb ANKA antigens Ig G1 and Ig G2 a did not change,especially serum antibodies specific to recombinant MSP119 antibodies also have no change.2.Effect of PD-1 elimination on Pb ANKA reinfection.In the reinfection,the normal infection rate of mice increased quickly,and the infection rate reached about 40% on the 18 th day after the reinfection,and then the mice began to die,and the PD-1 blocking caused the mouse erythrocyte infection rate to decrease obviously,and finally recovered,the mice all survived.On the 5th day after reinfection,the expression of splenic c DCs and p DCs,MHC?,CD86,and PD-L1 were detected by flow cytometry on DCs.In Pb ANKA infection,c DC and p DC increased significantly on the5 th day after infection,and the expression of CD86 and MHC? in DCs increased significantly after the elimination of PD-1,but there is no change of the expression of PD-L1.Effects of PD-1 blocking on the number of Tregs,MDSCs,Th1,activated T,Tfh in mouse splenocytes on day 5 after reinfection were detected by flow cytometry.Cytokines were detected by ELISA.We found that on the 5th day after reinfection,the elimination of PD-1 had no obvious effect on Treg,Tfh and Th1.MDSCs and activated T cells increased significantly.The IL-10,IL-21 and IL-6m RNA of spleen cells had no significant difference.Then the cytokines in the supernatant of spleen cell culture were detected.In reinfection,TNF-? and IL-6 increased significantly after PD-1 was eliminated,but IFN-? did not change significantly,and IL-10 decreased significantly.Effects of PD-1 blocking on the number of TCM,TEM,short-lived,long-lived plasma cells were detected by flow cytometry.Serum antibodies specific to Pb ANKA antigens Ig G1 and Ig G2 a and serum antibodies specific to recombinant MSP119 antibodies were detected by ELISA on day 5 after reinfection.We found that on the 5th day after infection,TCM increased significantly after PD-1 was eliminated,but TEM did not change.Although short-lived plasma cells did not change significantly,long-lived plasma cells increased significantly.Serum antibodies specific to Pb ANKA antigens Ig G1 and Ig G2 a increased after PD-1 elimination,especially serum antibodies specific to recombinant MSP119 antibodies increased after PD-1 elimination.PD-1 could significantly enhance the immune memory function of mice after elimination of PD-1.Conclusions 1.The innate and adaptive immune response were impaired in anti-PD-1m Ab-treated BALB/c mice after Pb ANKA infection as compared to control infected mice 2.The level of immune memory in BALB/c mice infected with Pb ANKA had been inhibited after PD-1 deletion during primary infection.3.The level of innate immune and adaptive response in BALB/c mice infected with Pb ANKA was significantly higher than that in PD-1 deletion during reinfection.4.The level of immune memory in BALB/c mice infected with Pb ANKA was significantly higher than that in PD-1 deletion during reinfection. |
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