Expression And Localization Of Plasmodium Berghei TIP And Its Immunomodulatory Effect In Experimental Cerebral Malaria

Objective:Cerebral malaria(CM)is the most serious complication of Plasmodium falciparum infection,characterized by central nervous system dysfunction,and kills nearly 400,000 African children every year.Although the exact molecular mechanism remains unclear,it is certain that CM occurs through a combination of factors,including mechanical vascular obstruction,cytokine dysregulation,chemokine production,and inflammation.Current studies suggested that the pathogenesis of CM is mainly related to the excessive activation of the immune system and the secretion of pro-inflammatory cytokines,such as TNF-α,IFN-γ,IL-1β,IL-6 and IL-12.These cytokines promote the expression of cerebral vascular endothelial cell adhesion molecules,leading to the sequestration of Plasmodium-infected erythrocytes in cerebral microvessels,and can also induce the production of chemokines to enhance the adhesion between CD8~+T cells and the vascular endothelium.Due to the accumulation of infected red blood cells and immune cells in the cerebral blood vessels,causing vascular obstruction,increased intravascular pressure,and increased permeability of the blood-brain barrier,cerebral edema is prone to occur,thereby inducing CM.Therefore,the regulation of T cell-mediated inflammatory response is crucial for suppressing immunopathological damage and the occurrence of CM.T cell immunomodulatory protein(TIP)is a protein discovered by Fiscella and other researchers that can inhibit the immune response of T cells.It has a protective effect in a mouse model of acute graft-versus-host disease and can significantly inhibit of the immune rejection caused by the graft,suggesting that TIP has the function of inhibiting the immune response of the body.Bioinformatics analysis found that there are proteins similar in structure to TIP in various malaria parasites.However,whether the Plasmodium TIP-like protein has high functional similarity with TIP,its expression and localization in different developmental stages of Plasmodium,and whether it can inhibit the host’s immune attack against Plasmodium remains unclear.Therefore,on the basis of the previous work,this project intends to study the expression and localization of P.berghei T cell immunomodulatory protein(PbTIP),and infects C57BL/6 mice with P.berghei ANKA to establish an experimental cerebral malaria(ECM)model,further to explore the role ofPbTIP in the development of CM,and strive to reveal the possible immune regulation mechanism,so as to provide a new theoretical basis and scientific basis for the effective prevention and treatment of malaria.Methods:1.Bioinformatics analysis ofPbTIP.BLAST was used for multiple sequence alignment,and MAGA 5 was used to analyze phylogenetic tree.SMART was used to predict protein domains,and Photoshop and DOG 2.0 software were used to draw pattern diagram.The online software(http://imed.med.ucm.es/)was used for analysis antigenic determinants.2.Expression and purification of rPbTIP.We selected the fragment(30-659aa)excluded transmembrane regions to express recombinantPbTIP(rPbTIP).The targeted fragment was amplified with primersPbTIP-F andPbTIP-R,then cloned into p ET28a vector,and the recombinant plasmid was transferred into E.coli BL21(DE3)cells,and induced at1.0 m M IPTG at 15℃for 16 h.After the recombinant protein was purified by Ni-NTA column,its concentration was determined by BCA method,and the purity and molecular weight of the protein were determined by 10%SDS-PAGE gel electrophoresis.3.Preparation of rPbTIP immune serum.Female BALB/c mice aged 6-8 weeks were subcutaneously injected with emulsified product of rPbTIP(50μg/mouse)and complete Freund’s adjuvant,and boosted rPbTIP(25μg/mouse)every two weeks.A total of two boosters were administered,and the control group was subcutaneously injected with PBS using the same immunization procedure.The mouse serum was collected before each immunization and 10 days after the last immunization,and the antibody titer of the corresponding serum was detected by ELISA.4.Detection ofPbTIP expression and localization:(1)The trophozoite,schizont,gametocyte and ookinete of P.berghei were isolated and purified using different Nycodenz gradients.Total RNA was extracted and reverse transcribed into c DNA,amplified by specific primers,and the m RNA expression ofPbTIP was detected by q PCR.(2)The purified Plasmodium was resuspended in lysis buffer containing 2%SDS and protease inhibitors,and the total protein was extracted.The rPbTIP immune serum was used to analyze the expression stage ofPbTIP by Western blot,and the serum of infected mice and the supernatant of ookinete culture were detected.(3)IFA was performed with rPbTIP immune serum to analyze the localization ofPbTIP in different stages of P.berghei,and images were acquired by confocal microscopy.5.Evaluation ofPbTIP’s transmission blocking activity.The rPbTIP immune serum or control serum was diluted 1:5,1:10 and 1:50 with ookinete medium,and mixed with 10μl mouse blood containing the same amount of mature gametocytes,then incubated at25℃for 15 min and 19℃for 24 h,respectively.Exflagellation centers and ookinetes were counted under the microscope.6.To analyze the functional activity of rPbTIP in vitro.Female 6-8-week-old C57BL/6mice were intraperitoneally injected with 1×10~6P.berghei-infected red blood cells.On day 3 post-infection,splenocytes were isolated and cultured.They were given different concentrations of rPbTIP(5,50,500 ng/ml),and heat-inactivated rPbTIP(50 ng/ml)was used as the control.The culture supernatant was collected after 48 h,and the levels of IFN-γ,TNF-α,IL-10 and TGF-βwere detected by ELISA.7.To analyze the effect ofPbTIP inhibition on the occurrence of CM in vivo.Female6-8-week-old C57BL/6 mice were intraperitoneally injected with 1×10~6P.berghei infected red blood cells to establish the ECM model.Anti-rPbTIP serum treated mice in the infection group(Pb A+anti-TIP)were injected with specific antibodies(100μl/mouse)in the tail vein before infection(day 0)and after infection(day 1,2).Pb A mice were given an equal amount of immune control serum at the same time.(1)The survival of mice,the time of occurrence of CM and symptoms were observed every day.The blood smears were prepared on day 3 post-infection,and parasitemia was counted under the microscope after Giemsa staining.(2)On day 8 post-infection,when the mice developed neurological symptoms,the mice from each group were randomly taken out,and the integrity of the blood-brain barrier was detected by evans blue(EB)staining.(3)On day 8 post-infection,when the mice developed neurological symptoms,the mice from each group were randomly taken out,and the brain tissue was fixed,embedded and sectioned for hematoxylin and eosin(HE)staining to observe the occlusion of brain microvessels and leukocyte infiltration.(4)On day 0,3 and 5 post-infection,the splenocytes were isolated,and flow cytometry was used to detect the proportion of Th1 cells,regulatory T cells(Tregs),dendritic cells(DCs)and their subpopulations.(5)On day 0,3 and 5 post-infection,the splenocytes were isolated,and the m RNA levels of IFN-γ,TNF-α,IL-10 and TGF-βwere detected by q PCR.(6)On day 0,3 and 5 post-infection,the splenocytes were isolated,and the supernatant was collected after 48 h.The expression levels of IFN-γ,TNF-α,IL-10 and TGF-βwere detected by ELISA.Results:1.Plasmo DB describesPbTIP as a"conserved Plasmodium protein,whose function is currently unknown".This gene(PBANKA＿1243600)is located on chromosome 12 and encodes a total of 703 amino acids with a molecular weight of 80.4 k Da.Prediction of protein domains showed thatPbTIP had two transmembrane regions at both N and C ends,and also contained a VCBS domain.The phylogenetic analysis showed thatPbTIP was relatively conserved in Plasmodium species,and was similar to human TIP structure.2.We selected the fragment of 630 amino acids to synthesize the targeted gene,and successfully constructed a prokaryotic expression vector p ET28a-PbTIP.After inducible expression and purification by affinity chromatography,high-purity rPbTIP was obtained.SDS-PAGE electrophoresis showed that targeted band was single and clear,consistent with the predicted molecular weight(～73 k Da),and the determined protein concentration was 0.96 mg/ml.3.The serum antibody titer of mice immunized with rPbTIP was significantly higher than that of the control group,indicating that rPbTIP has strong immunogenicity.4.Expression and localization ofPbTIP:(1)The q PCR results showed thatPbTIP m RNA was expressed in trophozoite,schizont,gametocyte and ookinete,and there was no significant difference in levels.(2)Western blot analysis of the purified Plasmodium protein extract showed that the rPbTIP antibody specifically recognized a band of about 80 k Da at trophozoite,schizont,gametocyte and ookinete,which was consistent with the predicted molecular weight.We also detected the presence of a small amount ofPbTIP in the serum of infected mice and the supernatant of ookinete culture,indicating thatPbTIP may be secreted outside the cell.(3)The IFA results showed thatPbTIP was evenly distributed in the cytoplasm of trophozoite,schizont,as well as male and female gametocyte,while there was almost no staining on the surface and cytoplasm of infected erythrocytes.In addition,PbTIP was mainly expressed on the membrane surface of male and female gametes and ookinete,suggesting thatPbTIP exists in different stages of P.berghei growth and development.5.PbTIP doesn’t have transmission blocking activity.Compared with the immune control serum,the anti-rPbTIP serum diluted at 1:5,1:10 and 1:50 could not effectively inhibit the formation of P.berghei male gametes and ookinetes.6.rPbTIP has immunomodulatory activity.Under the effect of three concentrations of rPbTIP,the expression levels of IFN-γand TNF-αin the supernatant of splenocytes decreased.The level of IL-10 increased at 500 ng/ml,and the levels of TGF-βwere significantly increased at 50 and 500 ng/ml,while heat-inactivated rPbTIP had no effect on the production of the aforementioned cytokines.7.The role and mechanism ofPbTIP in the occurrence of CM:(1)On day 9 post-infection,mice in thePb A group died,accompanied by neurological symptoms,and all died until day 11,while mice in thePb A+anti-TIP group died on day 8and 9 post-infection.Parasitemia of mice in thePb A+anti-TIP group was lower than that in thePb A group on day 7 post-infection.(2)The results of EB staining showed that compared with thePb A group,the blood-brain barrier of mice in thePb A+anti-TIP group was severely damaged,and there was obvious EB exudation.(3)The results of HE staining showed that the number of blood vessels infiltrated by leukocytes in the brain tissue of mice in thePb A+anti-TIP group was significantly higher than thePb A group.(4)Flow cytometry analysis showed that on day 3 and 5 post-infection,the proportion of Th1 cells in thePb A+anti-TIP group was higher than that in thePb A group.On day 3,the proportion of Tregs between the two groups was no significant difference.On day 5,the proportion of Tregs in thePb A+anti-TIP group was lower than thePb A group.On day 3,the proportions of DCs,m DCs and p DCs in thePb A+anti-TIP group were higher than those in thePb A group,and MHC II was highly expressed,but there was no significant change in these cells between the two groups on day 5.(5)The q PCR results showed that on day 3 post-infection,the m RNA levels of IFN-γand TNF-αin the splenocytes of mice in thePb A+anti-TIP group were higher than those in thePb A group.On day 5,the level of IFN-γwas higher than thePb A group.On day 3post-infection,there was no significant difference in the transcription levels of IL-10 and TGF-βbetween the two groups,while the levels of thePb A+anti-TIP group were lower than thePb A group on day 5.(6)ELISA results showed that on day 3 post-infection,the level of TNF-αin the culture supernatant of splenocytes in thePb A+anti-TIP group was significantly higher than that in thePb A group,and the level of IFN-γin thePb A+anti-TIP group was higher than thePb A group on day 5.There was no significant change in the levels of IL-10 and TGF-βbetween the two groups on day 3,and the IL-10 level in thePb A+anti-TIP group was lower than thePb A group on day 5.Conclusion:1.PbTIP is expressed in different stages of P.berghei growth and development,and can be secreted outside the erythrocytes in a soluble form.2.rPbTIP has immunomodulatory effects,which can down-regulate the expression of pro-inflammatory cytokines IFN-γand TNF-α,and up-regulate the expression of anti-inflammatory cytokines IL-10 and TGF-β.3.PbTIP is involved in the occurrence of CM.Inhibition ofPbTIP can shorten the survival period of mice and aggravate the symptoms of CM.4.PbTIP can regulate Th1 response.Inhibition ofPbTIP increases the proportion of Th1cells in mice,and up-regulates the expression of pro-inflammatory cytokines IFN-γand TNF-α.5.PbTIP can regulate Tregs response.Inhibition ofPbTIP reduces the proportion of Tregs in mice,and down-regulates the expression of anti-inflammatory cytokines IL-10and TGF-β.6.PbTIP is involved in the regulation of Th1 and Tregs immune responses by affecting the differentiation and maturation of DCs,thereby changing the process of CM.