Research Of Tregs Role On Regulating The Protective Immune Response And Immuneopathological Mechanisms In Malaria

Malaria infection is a major health problem to human beings even today.A recent survey has shown that,in 2002,roughly 2.2 billion people were at risk of P.falciparum infection,with a conservative estimate of 515 million individuals who had been infected.The prevention and therapy of such infectious diseases as Malaria,AIDS and tuberculosis have been taken as the focus of researches by WHO.However,Up till now, the mechanisms of immune response to malaria parasite infection are not fully understood yet.Thus,the rational development of effective anti-malarial vaccines and novel therapies to alleviate or prevent the symptoms of severe malaria infection requires a better understanding of the various mechanisms of immune responses to malaria parasite.A series of studies have proved that immune effector mechanisms are required to eliminate malaria parasites,and Th1 immune effectors are crucial to control the early infection.Experiments in vivo and in vitro have demonstrated:(1) CD4⁺ T cells are essential for the control of primary parasitemia in mouse models during the blood-stage of malaria infection.IFN-γplays an important role in the production and maintenance of Th1 in early response and control the outbreak proliferation of blood-stage malaria parasites.In addition,B cells secrete specific antibodies supporting by Th2 cells,which can effectively remove parasite,preventing the recidivation and recrudescence.Thus effective establishment of Th1 immune response and a successful switch to Th2 are crucial for educing protective immunity response in malaria.Balance and coordination of Th1/Th2 can not only control the crisis of the acute phase,but also help in the ultimate elimination of malaria parasite.(2) Phase and level of immune response might affect the final outcome of malaria infection.Related studies showed the serious complications of cerebral malaria were correlated with a strong Thl response.This indicated that it is critical for malaria infection to maintain the dynamic balance of inflammatory cytokines/anti-infiammatory cytokines and the control of inflammatory cytokine production time and intensity.Breaking or disturbing the balance and process of immune response,may be lead to chronic infection or the aggravation of pathogenetic condition.These observations also suggest the regulation of anti-malarial immune responses is as important as their induction in determining the final outcome of infection.However,the regulative mechanism on the dynamic balance of protective and pathologic immune response has not yet been clarified.CD4⁺CD25⁺ regulatory T Cells(Tregs) are a T cell subgroup that has immunoregulatory effects different from Th1 and Th2.Tregs are widely accepted as potent suppressors to play important roles in immunotolerance,autoimmunity,and transplantation.Tregs could suppress proliferation of many types of immunocytes including CD4⁺ T cells and CD8⁺ T cells and inhibit secretion of cytokines.Some data demonstrated that the disorder of immune function in host correlated with the chronically tendency of diseases.Reduction or depletion of Tregs could enhance anti-infection immunity to a variety of pathogens such as bacteria,viruses,fungi and parasites.But the study on the role of Tregs in malaria and other infectious disease has just start.In addition,people found that the transcription factor also has an important role during the cell subsets differentiation process.T-bet and GATA3 are two kinds of transcription factors within cells,specific expression on the surface of Th1/Th2 cells respectively.T-bet regulates the development of Th1 cells,GATA3 regulates the development of Th2 cells,and thus T-bet and GATA3 are the crucial factors that Th0 transform to Th1/Th2. In present study we investigated the relation between the different of Th1/Th2 immune responses and effects of Tregs.Therefore,using rodent model of BALB/c and DBA/2 mice infected with P.y17XL,P.cAS and P.y17XL plus P.cAS,respectively.Then we determined the number of Tregs and level of cytokines as,IFN-γ,IL-4,IL-10 and TGF-βand percentage of apoptotic CD4⁺ T cells,to investigate the role and the possible immunosuppressive mechanisms of Tregs.On the basis of it,in vivo CD25 depletion was performed to further determine the role and mechanisms of Tregs in early stage of malaria infection.Methods1.Experimental animal and models of constructionFemale 6-8-weeks-old BALB/c and DBA/2 mice were infections with 1×10⁶ Py17XL,P.cAS and 2×10⁶ P.y17XL plus P.cAS(1:1) parasitized erythrocytes(PE) by intraperitoneal(i.p.),Constructed different experimental animal models.2.Determination of contents of IFN-γand IL-4 in malaria parasite infected DBA/2 and BALB/c mice by double antibody sandwich ELISASpleen cells were harvested from mice and adjusted to a final concentration of 10⁷cells/ml.Aliquots(500μl/well) of the cell suspension were incubated in 24-well plat-bottom tissue culture plates(FALCON) in triplicate for 48 hours at 37℃in a humidified 5%CO₂ incubator.Then the 24-well plates were centrifuged at 350g for 10 min at RT,supernatants were collected and stored at-80℃until the assayed for IFN-γand IL-4 levels.3.Detection of transcription factors T-bet and GATA3 mRNA expression level by RT-PCR(1) PCR primerUsing primer design software Primer5 design T-bet,GATA3 andβ-actin specific primers. (2)RNA extractionUsing TRIZOL one-step method extract to total RNA in spleen cells of mice and the UV spectrophotometer measured purity,the ratio of RNAA₂₆₀/A₂₈₀ is between 1.8 and 2.0.4.Determination by flow cytometry of the proportion of Tregs and apoptotic CD4⁺ T cells in malaria parasite infected DBA/2 and BALB/c miceSpleen cells were aliquoted into staining tubes at approximately 1×10⁶ cells per tube and incubated with Mouse-block to block non-specific binding of fluorochrome-labeled antibodies.Two-color staining was then performed using FITC-labeled anti-mouse CD4 mAb and PE-labeled anti-mouse CD25 mAb simultaneously;The antibodies were diluted in FASC buffer and incubated with the cells for 30 min on ice without light.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest programme.Spleen cells diluted with binding buffer were aliquoted into staining tubes at approximately 1×10⁵ cells per tube.Three-color staining was then performed using FITC-labeled anti-mouse CD4 mAb,PE-labeled anti-mouse Annexin V and 7AAD simultaneously,and incubated with the cells for 15 min at 25℃without light.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest programmeFlow cytometric analysis utilized linear forward light scatter(FS),linear side light scatter(SS) and log fluorescence parameters.5.Determination of the contents of IL-10 and TGF-βin malaria parasite infected DBA/2 and BALB/c mice by double antibody sandwich ELISAAll cells used were spleen cells harvested from mice and concentration of cells were adjusted to 1×10⁷/ml and 500μl cells per well were plated on the plastic 24-well plates with RPMI 1640 medium supplemented with 10%heat-inactivated FCS in 95% air-5%CO₂ at 37℃for 48 hours.Supernatants were collected after centrifugation at room temperature,350g.Supernatants above were tested in duplicate using ELISA kits for IL-10 and TGF-β.6.Intracytoplasmic stainingSpleen cells from BALB/c and DBA/2 mice were collected at different time point after infection.After stimulated for 2 hours with PMA and ionomycin at 37℃,Golgi Stop was added to each reaction(1:500,vol/vol).After continued co-culture for 4 hours at 37℃,cells were washed with 3%FCS and then resuspended in 100μl of 3%FCS. FITC-anti-CD4 and PE-anti-CD25 were added for surface staining.Then cells were fixed and permeabilized,and intracytoplasmic staining was performed using allophycocyanin(APC)-anti-IL-10.FITC rat IgG2b was used as the isotype control.7.CD25 depletion in vivoDepletion of Tregs in BALB/c mice was carried out respectively by i.p. administration of 1mg anti-mouse CD25 mAb(7D4) 1 day before parasite challenge and on day 1 pi.Control group was carried out by i.p.administration of PBS.Tregs depletion was confirmed by flow cytometry.Spleen cell cultures harvested from Py17XL-infected BALB/c mice on days 0,3 and 5 pi was used to analyze level of cytokines IFN-γand IL-10 and FCSA were performed to determine the change number of Tregs and apoptosic CD4⁺ T cells after in vivo depletion of Tregs.Results1.Comparison of the concentration of IFN-γand IL-4 from spleen supernatants in malaria parasite infected DBA/2 and BALB/c miceThe level of IFN-γincreased rapidly and reached the peak on day 3 pi and then decreased slowly in P.y17XL-infected DBA/2 mice,while in BALB/c mice,the level of IFN-γonly increased significantly on day 3 pi,which was followed by a sharp reduction.In contrast,the level of IFN-γonly transient increased significantly on day 3 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice,the level of IFN-γincreased significantly on day 3 pi,and peaked on day 5 pi;The level of IFN-γreached peak on day 3 pi in mixed-species infected DBA/2 and BALB/c mice.The level of IL-4 only increased significantly on day 3 pi in P.y17XL-infected BALB/c mice,while in DBA/2 mice,the level of IL-4 increased and reached the peak on day 5-8 pi,then reduction slowly.In contrast,the level of IL-4 only transient increased significantly on day 5 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice,the level of IL-4 increased and peaked on day 8-10 pi,followed decreased,but still higher than normal.There was no notable change of IL-4 on day 1-5 pi in mixed-species infected DBA/2 and BALB/c mice,and peaked on day 8 pi,then decreased down to normal.2.Comparison of the expression and the ratio of T-bet and GATA3 from spleen cells in malaria parasite infected DBA/2 and BALB/c miceThe expression of T-bet mRNA in both P.y17XL and P.cAS infected DBA/2 mice increased and was higher than that of GATA3,the ratio of T-bet/GATA3 increased significantly in early phase of infection.In contrast,the expression of T-bet and GATA3 mRNA in both P.y17XL and P.cAS infected BALB/c mice have no obviously change on day 3-5 pi,the ratio of T-bet/GATA3 increased significantly on day 1 and 3 pi respectively.3.Comparison of differences in proportion of Tregs between malaria parasite infected DBA/2 and BALB/c miceThe percentage of Tregs raised significantly on day 1 pi in P.y17XL-infected mice, and the percentage of Tregs increased slowly,peaked on day 5 pi and then decreased; while the percentage of Tregs increased rapidly and reached the peak on day 5 pi.In contrast,the percentage of Tregs reached the peak on day 5 pi and 10 pi in P.cAS-infected DBA/2 and BALB/c mice respectively.The percentage of Tregs increased slowly and peaked on day 5 pi,followed decreased in mixed-infected DBA/2 mice,while the percentage of Tregs rapidly raise and reached the peak on day 8 pi.4.Comparison of differences in proportion of CD4⁺ T cells between malaria parasite infected DBA/2 and BALB/c miceThe percentage of apoptotic CD4⁺ T cells increased slowly in P.y17XL-infected DBA/2 mice,while the percentage of apoptotic CD4⁺ T cells increased rapidly on day 3-5 pi in BALB/c mice;In contrast,the percentage of apoptotic CD4⁺T cells raised significantly only on day 8 pi in P.cAS-infected BALB/c mice,while in DBA/2 mice, the percentage of apoptotic CD4⁺T cells reached the peak on day 8 pi.5.Comparison of the contents of IL-10 and TGF-βin malaria parasite infected DBA/2 and BALB/c miceThe concentration of IL-10 increased significantly and kept an elevated level from day 1 to day 5 in P.y17XL-infected DBA/2 and BALB/c mice.In contrast,the level of IL-10 reached the peak on day 8 pi and 10 pi in P.cAS-infected BALB/c and DBA/2 mice.The level of TGF-βraised significantly on day 10 pi and 12 pi in P.y17XL -infected DBA/2 mice and then decreased down to normal;while in BALB/c mice,the level of TGF-βincreased significantly only on day 5 pi.In contrast,the level of TGF-βraised and peaked on day 8 pi in P.cAS-infected DBA/2 mice,while in BALB/c mice, the level of TGF-βtransient increased significantly on day 3 pi and then decreased down to normal on day 8 pi.6.The change of immune response mode in BALB/c mice during the early stage of P.y17XL infection after CD25 depletion in vivoIn CD25-depleted BALB/c mice,parasitemia increased slowly and reached the low-level peak,survival rate was obviously prolonged,and even part of CD25-depleted mice survival;The level of IFN-γmarkedly elevated,while the level of IL-10 markedly decreased.Conclusions1.Immune response mode is different in DBA/2 and BALB/c mice during P.y17XL and P.cAS infection respectively.2.It is essential for the establishment of effective Th1 and Th2 responses and a successful switch of immune response from Th1 to Th2 in DBA/2 and BALB/c mice during P.y17XL and P.cAS infection respectively.And phase and level of immune response might affect the final outcome of malaria infection.3.The increase of Tregs amount in P.y17XL-infected BALB/c mice is closely correlated with failure to establishment and maintenance of Th1 response in the early stage of infection.4.The abnormal activation of Tregs is related with failure to switch of immune response from Th1 to Th2 in P.cAS-infected DBA/2 mice.5.The immune response mode in mixed-species infected DBA/2 and BALB/c mice is the same as that of P.y17XL-infected mice.6.The protective immunity against mixed-species malaria infections depends on the establishment of effective Th1 and Th2 responses and a successful switch of immune response from Th1 to Th2.7.The persistent increase of Tregs amount is correlated with failure to establishment and maintenance of Th1 response in mixed-species infected BALB/c mice,and seemed to significantly affect the final outcome of malaria infection through its regulation of the Th1 and Th2 response.8.Tregs have a crucial role to regulate Th1 responses,potential regulatory mechanisms include IL-10-dependent manner and induce CD4⁺T cells apoptosis during the early stage of P.y17XL infection.