Research On The Role Of Dendritic Cells During The Early Stage Of Plasmodium Yoelii Infection

ObjectiveMalaria,caused by the intracellular parasite Plasmodium,is the most important parasitic infection in humans.The global prevalence of malaria continues to rise at an alarming rate and is a major cause of high mortality in children and morbidity in infected adults living in the developing world.Despite extensive research,a successful vaccine is not yet available and many antimalarial drugs are increasingly ineffective due to widespread drug resistance.Therefore,a better understanding of the mechanisms that induce protective immunity to malaria infection will provide necessary and primary data for effective control malaria.Mice infected with rodent malaria parasites may provide a valuable animal model for dissecting protective immunity against human malaria.Different profiles of Th1 immune responses have been observed in different strains of mice infected with P.yoelii 17XL,which lead to distinct consequences-spontaneous cure and death, indicating that effective Th1 immune response in the early stages of infection critically influences the later development and final outcome of malaria infection.However,the factors responsible for regulation of Th1 immune responses for host resistance to blood-stage malaria are poorly understood.Dendritic cells(DCs),which express distinct pattern recognition receptors(PRRs) such as Toll-like receptors(TLRs),play a crucial role in the activation of T cells and consequently in the induction of adaptive immune responses.It is well established that DCs are divided into different subpopulations:myeloid DCs(CD11c⁺CD11b⁺) and plasmacytoid DCs(CD11c⁺CD45R/B220⁺).They usually exhibit distinct patterns of proliferation by different pathogen stimuli.A recent study found that DCs selectively phagocytose plasmodium-parasitized RBCs(pRBCs) and present pRBCs-derived Ags to CD4⁺T cells in vitro,indicating that DCs may play a primary role as APCs in malaria infection,while other findings suggested that the functions of DCs were suppressed by malaria parasites.We have previously established a rodent model for analysis the factors involved in the regulation of immune response induced by Plasmodium infection by demonstrating that DBA/2 mice were resistant to and BALB/c mice were susceptible to P.yoelii 17XL infection,due to different ability to induce Th1 immune responses.To investigate whether DCs are implicated in the establishment and regulation of Th1 immune responses during the early infection of P.yoelii 17XL.In this study,we utilized P.yoelii 17XL infected DBA/2 and BALB/c mouse strains,and then determined the levels of cell surface TLR4 and intracellular TLR9 of DCs and the numbers of CD11c⁺CD11b⁺ DCs and CD11c⁺CD45R/B220⁺ DCs,to investigate the activation and multiplication characteristics of DCs.On the basis of it,the expression levels of three kinds of maturation markers(MHCâ…¡,CD40 and CD80) and the levels of cytokines as IL-12p40,IL-10 and TGF-β1 were analyzed to further determine the mechanisms of DCs in the early stage of malaria infection.Method1.Experimental animal and models of constructionFemale 6-8-weeks-old BALB/c and DBA/2 mice were infections with 1×10⁶ P.yoelii 17XL parasitized RBC(pRBC) by intraperitoneal(i.p.),Constructed different experimental animal models.2.Determination by flow cytometry of the levels of TLR9/4 expression on DCs in BALB/c and DBA/2 mice during the early stage of infection with P.yoelii 17XLFor DCs cell surface TLR4 analysis,splenocytes were harvested from mice and adjusted to a final concentration of 1×10⁷ cells/ml,blocked with anti-CD16/CD32,and then stained using a combination of FITC anti-CD11c mAb(HL3) and PE anti-TLR4 mAb(MTS510).For intracellular TLR9 staining of DCs,splenocytes were blocked with anti-CD16/CD32 after harvesting,and then stained using FITC anti-CD11c mAb(HL3). After fixation and permeabilization using staining buffer reagents as instructed by the manufacturer,cells were incubated with biotinylated anti-TLR9 mAb(5G5,Hbt) followed by PE-conjugated streptavidin.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest program.3.Determination by flow cytometry of the percentage of DC subpopulations and DCs expressing MHCâ…¡,CD40,CD80 in BALB/c and DBA/2 mice during the early stage of infection with P.yoelii 17XL(1) Spleen cells were aliquoted into staining tubes at approximately 1×10⁶ cells per tube and incubated with anti-CD16/CD32 to block non-specific binding of fluorochrome-labeled antibodies.Three-color staining was then performed using FITC-labeled anti-mouse CD11c mAb and PE-labeled anti-mouse CD11b mAb and PerCP-labeled anti-mouse CD45R/B220 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 program.(2) Two-color staining was then performed using FITC-labeled anti-mouse CD11c mAb and PE-labeled anti-mouse MHCâ…¡mAb,PE-labeled anti-mouse CD40 mAb or PE-labeled anti-mouse CD80 mAb respectively,and incubated with the cells for 30 min on ice without light.FACS analyses were performed on a FACSCalibur instrument run with the CELLQuest program.4.Determination of the contents of IL-12p40,IL-10 and TGF-β1 from DC culture supernatants in BALB/c and DBA/2 mice by double antibody sandwich ELISA during the early stage of infection with P.yoelii 17XL(1) Spleens from na(l|¨)ve or infected mice were treated for 30 min at 37℃with 1 mg/ml collagenaseâ…£(Sigma Aldrich).The spleens were then homogenized into single-cell suspension,splenic DCs were isolated using BD IMag^TM Mouse Dendritic Cell Enrichment set-DM(Cat.no.557955) according to manufactures instructions.The BD IMag^TM Mouse Dendritic Cell Enrichment Set-DM was used for the negative selection of DCs from mouse spleen.The Biotinylated Mouse Dendritic Cell Enrichment Cocktail contain monoclonal antibodies that recognize antigens expressed on peripheral erythrocytes and leukocytes that are not DCs.The BD IMag^TM Streptavidin particles plus-DM are magnetic nanoparticles that have streptavidin covalently conjugated to their surfaces.With these two components,the BD IMag^TM Mouse Dendritic Cell Enrichment set-DM avoids the inadvertent activation of the enriched DCs by using reagents that do not directly bind to those DCs.Briefly,Cells (20×10⁶) were Fc blocked with 2.4G2 mAb(BD Pharmingen San Diego,CA) and labeled with biotinylated mouse dendritic cell enrichment cocktail at 5μl per 1×10⁶ cells(BD Pharmingen San Diego,CA).Then added 5μ1 BD IMag^TM Streptavidin particles Plus-DM for every 1×10⁶ cells,transferred the cells to a 17×100mm round-bottom test tube(Cat.no.352057) and placed the positive-fraction tube on the BD IMagnet^TM.The purified DCs were routinely 70～85%CD11c⁺,as determined by flow cytometry.Viability was assessed by trypan blue dye or labeled with 7-actinomycin D.Aliquots of 1 ml DC suspensions at a concentration of 1×10⁶ cells/ml were cultured in triplicate in 24-well tissue culture plates in complete medium.The cultures of splenic DCs(5×10⁵ cells/well) were incubated for 48 h at 37℃in a humidified CO₂ incubator.Supernatants were collected and stored at-80℃until assayed for cytokine levels.(2) Levels of IL-12p40,IL-10 and TGF-β1 from DC culture supernatants were 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 IL-12p40,IL-10 and TGF-β1 in samples were calculated against the standard curve generated using recombinant cytokine.Results1.Comparison of TLRg/4 expression on DCs in BALB/c and DBA/2 mice during the early stage of infection with P.yoelii 17XLThe population of CD11c⁺ DCs expressing TLR9 significantly increased on day 3 and peaked on day 5 p.i.in BALB/c and DBA/2 mice.However,there was no statistical significance between two mice.Meanwhile,there was no change about the population of CD11c⁺ DCs expressing TLR4 in BALB/c and DBA/2 mice.2.Comparison of DC subpopulations in BALB/c and DBA/2 mice during the early stage of infection with P.yoelii 17XLThe population of splenic CD11c⁺CD11b⁺ DCs significantly increased on day 3 p.i.and peaked on day 5 p.i.in DBA/2 mice,whereas the percentage of CD11c⁺ CD11b⁺ DCs in BALB/c mice lowered that in DBA/2 mice on day 3 p.i. Although,the proportion of splenic CD11c⁺CD45R/B220⁺ DCs in BALB/c and DBA/2 mice showed gradually increasing trend from day 3 to day 5 p.i.,BALB/c mice showed the proportion of splenic CD11c⁺CD45R/B220⁺ DCs was significantly higher than those in DBA/2 mice from day 3 p.i.to day 5 p.i.3.Comparison of MHCâ…¡,CD40 and CD80 expression on DCs in BALB/c and DBA/2 mice during the early stage of infection with P.yoelii 17XLThe population of DCs expressing MHCâ…¡and CD40 significantly increased on day 3 and peaked on 5 p.i.in BALB/c and DBA/2 mice.However,the levels of BALB/c mice were less than those of DBA/2 mice.Meanwhile,the population of DCs expressing CD80 in DBA/2 mice obviously increased on day 3 and peaked on day 5 p.i., which were higher than those in BALB/c mice.Nevertheless,the percentage of DCs expressing CD80 in BALB/c mice significantly increased only on day 5 p.i.4.Comparison of the contents of IL-12p40,IL-10 and TGF-β1 in DBA/2 and BALB/c mice during the early stage of infection with P.yoelii 17XLThe levels of IL-12p40 in DC supernatants showed obvious increases on day 3 and 5 p.i.in DBA/2 mice,which was statistical significance compared with the pre-infection.However,a continuously increased levels of IL-12p40 in DC supernatants were not observed from day 3 to 5 p.i.in BALB/c mice.The levels of IL-10 and TGF-β1 in DC supernatants showed obvious increases on day 3 and 5 p.i.in BALB/c mice and DBA/2 mice.But the levels of BALB/c mice were higher than those of DBA/2 mice.Conclusions1.During the early infection of P.yoelii 17XL,TLR9 contributed to DC activation between susceptible and resistant mouse strains.2.During the early infection of P.yoelii 17XL,there are significant differences in DC subpopulations proliferation model in BALB/c and DBA/2 mice.3.During the early infection of P.yoelii 17XL,there are significant differences in the levels and phase of characteristic molecules MHCâ…¡,CD40 and CD80 reflecting mature phenotype in BALB/c and DBA/2 mice.4.During the early infection of Pyoelii 17XL,DCs can induce Th1 immune response development by secreting IL-12.5.During the early infection of P.yoelii 17XL,DCs can regulate the level and intensity of Th1 immune response by secreting immunoregulatory cytokines IL-10 and TGF-β1.6.During the early infection of P.yoelii 17XL,DCs are one of the crucial immune effector cells responsible for Th1 immune response establishment.