Research On Regulation Role Of CD4~+ CD25~+ Regulatory T Cells In Plasmodium Chabaudi Infection

ObjectiveMalaria is the most serious parasite disease transmitted by Anopheles mosquito vectors.The malaria epidemic areas distribute over 100 countries in the world and over 22 billion people are at risk of infection.Malaria kills over 110～270 million people and 350-550 million clinical cases are reported annually.The vaccines for malaria, together with HIV/AIDS and TB are urgently needed.Thus,it is an emphasis project that to develop effective malaria vaccines.Whereas the important premise found is the systemic basic research for immunology and molecular biology on the immune response of host against malaria parasite infection.Models of rodent malaria have proved that preventive immunity during intra-erythrocyte stage is set up with the involvement of CD4⁺ T cells and some antibodies.Successive activation of Th1-Th2 is the key to preventive immunity,and IFN-γplays an important role in the production and maintenance of Th1 in early response.Previous studies have shown that Th1 response is essential for resistant mice to clear parasites during intra-erythrocyte stage.IFN-γ,as an important regulatory Th1 cytokine,can activate such immunocytes as macrophages which can secrete non-specific effector molecules such as NO.CD4⁺CD25⁺ regulatory T cells(Tregs)have special regulation function which first reported by Sakaguchi on 1995.Different with CD4⁺ Th1/Th2 cells,Tregs specially expressed transcription factor Forkhead box P3(FoxP3)and surface molecular CD25, tumor necrosis factor receptor(GITR).FoxP3 has key role on development of Tregs. CD25,CTLA-4 and GITR are associated with Tregs function.The in vivo and intro researches showed that Tregs have general immune inhibit function.Recently,it is reported that depletion of Tregs could protect mice from death during a lethal strain of Plasmodium yoelii(Plasmodium yoelii17XL,P.yoelii 17XL)infection,and this protection was associated with specific T cell respones,suggesting that Tregs might contribute to immune suppression during malaria infection.In our previous study,we found that Tregs might be one of the mechanisms that Th1 cell immunity fail to be set up in BALB/c mice during early infection of lethal strain of P.yoelii 17XL.In contrast,BALB/c and DBA/2 mice had different infection outcome during Plasmodium chabaudi chabaudi AS(P.c.chabaudi AS).To investigate characteristic of Tregs activation and correlation with disease progression,BALB/c and DBA/2 mice were infected with P.c.chabaudi AS,and then determined the numbers of Tregs by FACS and levels of cytokines as IFN-γand IL-4 by ELISA,to investigate the role and the possible immunoregulation mechanisms of Tregs.Materials and methodsBALB/c and DBA/2 mice were injected intraperitoneal(i.p.)with 10⁶ blood stage of lethal strain of P.c.chabaudi AS.Parasitemia was monitored by microscopic evaluation of thin blood films with Giemsa staining.All cells used in experiments were spleen cells harvested from mice on 0,1,3,5,8,10,11,12 and 15 days post-infection, 1×10⁷ cells per well on the plastic 24-well plates with RPMI 1640 medium supplemented with 10%heat-inactivated FCS in 5%CO₂ at 37℃for 48 hours. Supernatants were collected and were tested in duplicate using ELISA kits for IFN_γ, and IL-4 according to the manufacturer's instructions.A single-cell suspensions with a number of 1×10⁶ cells were used for flow cytometry staining.Two-color staining was then performed using FITC-labeled anti-mouse CD4 mAb and PE-labeled anti-mouse CD25 mAb simultaneously.Percentage of Tregs in total spleen CD4⁺T cell population was measured by flow cytometry.Statistical significance of differences was analyzed by Student's t test.A value of P＜0.05 was considered significant.ResultsBALB/c mice infected with P.c.chabaudi AS developed a moderate parasitemia with a peak 34.4%on day 8 post inoculation(p.i.)following by a sharp decline and all mice survived on day 15 p.i..In addition,in cultured splenocyte supernatants from BALB/c,both IFN-γproduction and Tregs percentage increased significantly on day 5 p.i(P＜0.05)and then decreased.IL-4 production increased significantly on day 8 p.i (P＜0.05)and reached peak on day 10 p.i..However,DBA/2 mice infected with P.c. chabaudi AS showed a parasitemia which maintained approximately 38%by day 9-11d p.i.and all mice died around day 10 p.i..IFN-γproduction from splenocyte supernatants from DBA/2 increased significantly on day 3 p.i.(P＜0.01)and then dropped down quickly.IL-4 production increased significantly only on day 5 p.i.(P＜0.05).The percentage of Tregs from splenocyte supernatants from DBA/2 kept steady elevated level before day 8 p.i.(P＜0.01),while suddenly rose up by day 8-11 p.i.(P＜0.01).DiscussionModels of rodent malaria have proved that different mice that suffer from the same parasite infection have different infection outcome.In our study,we found that during P.c.chabaudi AS infection,BALB/c and DBA/2 had different infection outcome. BALB/c could survive when suffer from P.c.chabaudi AS infection while DBA/2 would die.Both BALB/c and DBA/2 would establish Th1 immune response represented IFN-γsecreted followed by Th2 immune response represented secreted IL-4.Production and successive activation of Th1-Th2 were the key to protect BALB/c from suffering from P.c.chabaudi AS infection.The strong Th1 immune response and failure to induce Th2 immune response were the reason for DBA/2 die.Thus,the essential factor for protecting mice from die is the establishment and successive activation mode of Th1-Th2 immune responses which is influenced by the activation time and response level of Tregs. Tregs are important effect cells in maintain normal immune balance and inhibit autoimmune disease.Tregs play inhibit role on activation and proliferation of many cells in vivo.Mendez S reported that Leishmania major's infection result in tissue injure aggravation is correlate obviously with number increase of Tregs in local tissues. Disorder in balance of Tregs and effect cells is major factor of disease aggravation. Depletion of Tregs could prevent from relapse.We found that DBA/2 was resistant to P. yoelii 17XL but susceptible to P.c.chabaudi AS,accompaniment with the Tregs number changed.The number of Tregs changed happened in the transition period of Th1 to Th2.Therefore,it suggested that Tregs was associated with disease severity during P.c.chabaudi AS infection.In our previous study,we found that Tregs might be one of the mechanisms that Th1 cell immunity fail to be set up in BALB/c mice during early infection of lethal strain of P.yoelii 17XL.In this study,our results suggested Tregs might play immune regulation role on the transition period of Th1 to Th2.The might mechanisms needed to be further studied.Conclusion1.During infection of lethal strain of P.c.chabaudi AS,Th1 and Th2 immune responses in BALB/c and DBA,/2 mice are different.2.The establishment and successive activation of Th1-Th2 are the key to preventive immunity.3.During infection of lethal strain of P.c.chabaudi AS,the changes of number and activation time of Tregs are closely associated with malaria severity.