Natural Regulatory T Cells Mediate The Development Of Cerebral Malaria By Modifying The Pro-inflammatory Response

Malaria continues to be a major health problem for humans. Malaria kills greater than 1 million children each year. Cerebral malaria (CM) is one of the most severe complications and is a major cause of death. The factors that determine disease severity are not completely understood, but are likely to include both parasite and host components. Ultimately, the interplay between the parasite and the immune response likely determines the outcome of the infection. Thus, fundamental research about the different underlying immune mechanisms of CM is the premise of developing effective anti-malaria vaccines and drugs.Plasmodium berghei ANKA (P.bANKA) murine malaria has many features in common with human disease and is thus the best available model for certain important aspects of clinical malaria. Current views support the notion the CM results from the combined effect of sequestration of parasitized red blood cells within blood vessels in the brain and a strong host pro-inflammatory response mediated by cytokines and effector cells. Corollary studies suggest that immune responses mediated by activated T cells are involved in disease induction. Specifically, both CD8+ and CD4+ subsets of T cells have been shown to contribute to cerebral pathogenesis. In fact, Thl responses appear to play a critical role in the control of parasitemia, and they also contribute to CM.CD4+CD25+ regulatory T Cells (Tregs) is a T cell subgroup that has different immunoregulatory effects than Thl and Th2. Activation of Tregs could constitute a mechanism utilized by the parasite to evade host immunity. In a previous study, we have shown that activation of Tregs is correlated with susceptibility of P. yoelii 17XL (P.yl7XL)-infected mice. Tregs can regulate the Thl response by modifying dendritic cells (DCs), the induction of IL-10, and apoptosis of CD4+ T cells. Therefore, in the current study we compared the dynamics of pro-and anti-inflammatory cytokines and Tregs proliferation in normal infected mice and Tregs-depleted mice infected with P. berghei to evaluate the role of Tregs in CM and the potential regulatory mechanisms.Method1. Construction of experimental animal models and disease assessmentCM-susceptible animal model was constructed by intraperitoneal (i.p.) injection of 1×106 P. berghei ANKA parasitized erythrocytes for C57BL/6 mice; CM-resistant animal model was constructed by intraperitoneal (i.p.) injection of 1×106 P. berghei ANKA parasitized erythrocytes for BALB/c and DBA/2 mice. Depletion of Tregs in C57BL/6, BALB/c and DBA/2 mice was carried out by i.p. administration of lmg of anti-mouse CD25 mAb (7D4, rat IgM, Bio Express) one day before parasite challenge and on day 1 p.i.. Control mice were treated with the same volume of PBS intraperitoneally. Mice were monitored twice daily after day 6 post infection (p.i.), and clinical experiment cerebral malaria (ECM) evaluated. Clinical ECM scores were defined by the presentation of the following signs:ruffled fur, hunching, wobbly gait, limb paralysis, convulsions and coma. Each sign was given a score of 1. Animals whose accumulative scores≥4 were considered to be with severe ECM.2. Determination of the percentage of Tregs in total spleen CD4+T cell population by flow cytometryThe spleen was aseptically removed from each mouse sacrificed on days 0,3,5,8 p.i.. Erythrocytes were lysed with cold 0.17 M NH4Cl. Spleen cells were adjusted to a final concentration of 107 cells/ml in RPMI-1640 supplemented with 10% heat-inactivated FCS. samples were triple stained with FITC-conjugated anti-mouse CD4/L3T4 (GK1.5), APC-conjugated anti-mouse CD25/IL-2 receptor alpha (PC61) and PE-conjugated anti-Foxp3 (clone FJK16s) (anti-Foxp3 antibody was from eBioscience, other antibodies were all from BD Pharmingen). Spleen cells were aliquoted into staining tubes at approximately 1×106 cells per tube and incubated with anti-CD 16/CD32 to block non-specific binding of fluorochrome-labeled antibodies. FITC-anti-CD4 and APC-anti-CD25 were added for surface staining (Abs were purchased from BD Pharmingen). Then cells were fixed and permeabilized, and intracytoplasmic staining was performed using PE-conjugated anti-Foxp3. After washing twice with PBS containing 1% FCS and suspended in 500μl of PBS, the cells were then analyzed in FACS Calibur Flow Cytometer (BD Bioscience) with Cell Quest software. Viable cells were gated by forward and side scattering. The percentage of Tregs in CD4+T cells was analyzed.3. Determination of the contents of IFN-γ, TNF-α, IL-6, IL-17, IL-10 from splenocyte culture supernatants by double antibody sandwich ELISA and determination of the contents of NO by Griess reaction(1) The preparation of spleen cell samples The spleen was aseptically removed from each mouse sacrificed on days 0,3,5,8 p.i.. Erythrocytes were lysed with cold 0.17M NH4Cl. Spleen cells were adjusted to a final concentration of 107 cells/ml in RPMI-1640 supplemented with 10% heat-inactivated FCS. Aliquots (500μ1/well) of the cell suspension were incubated in 24-well flat bottom culture plates (FALCON) in triplicate for 48 h at 37℃in a humidified 5% CO2 incubator. Specially naive spleen cells were cultured with purified NA/LE hamster anti-mouse CD3e (145-2C11, BD Pharmingen,0.56μl/well) and purified NA/LE hamster anti-mouse CD28 (37.51, BD Pharmingen,0.112μl/well) for 2 days at 37℃for stimulating the production of IL-17. Then the 24-well plates were centrifuged at 350 g for 10 min at RT, supernatants were collected and stored at-80℃until they were assayed for cytokine levels.(2) IFN-y, TNF-a, IL-6, IL-10, IL-17 levels in splenocyte supernatants were measured by commercial enzyme-linked 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 IFN-y, TNF-a, IL-6, IL-10 and IL-17 in samples were calculated against the standard curve generated using recombinant cytokines.(3) NO2- concentration in culture supernatant of cells was assayed by the Griess reaction. Briefly,100μl Griess reagent [equal volumes of 1%(w/v) sulfanilamide (Wako, Osaka, Japan) and 0.1%(w/v) N-1-naphtyl ethylenediamine dihydrochloride (Wako, Osaka, Japan) in 2.5%(w/v) H3PO4] for 10 min at RT, and the OD at 550nm was measured with a microplate reader. NO2- concentration was determined using NaNO2 as a standard.Results1. Parasitemia, survival rates and disease assessmentCM was induced in susceptible strains of C57BL/6 mice by injection of virulent P.bANKA blood-stage parasites. The C57BL/6 mice died after paralysis and coma ensued between days 8 and 11 after infectious challenge, with parasitemia between 10% and 20%. In contrast, resistant strains of BALB/c and DBA/2 suffered similar parasite growth, but no cerebral disease. They died 3-4 weeks after infection, with anemia and a parasite burden> 60%. CM-susceptible C57BL/6 mice were shown to score higher than CM-resistant BALB/c and DBA/2 mice based on the clinical scores used for assessment of the clinical symptoms of ECM, which indicated that cerebral pathology was more severe in the susceptible than resistant strains.2. Dynamics of pro-and anti-inflammatory cytokines and NO levels in C57BL/6, DBA/2, and BALB/c mice with P.bANKA infections The pro-inflammatory cytokines (IFN-γ, TNF-α, IL-6, and IL-17) and NO in the 3 mouse strains began to increase after infection with a peak on day 5 p.i. and a decline on day 8 p.i. (a slight decline in C57BL/6 mice) during the course of P.bANKA infection. The anti-inflammatory cytokine (IL-10) also reached a peak on day 5 p.i. following a significant decrease in C57BL/6 mice and a slight decrease in BALB/c and DBA/2 mice on day 8 p.i.. The levels of pro-inflammatory cytokines in CM-susceptible C57BL/6 mice were higher than in CM-resistant BALB/c and DBA/2 mice; IL-10 exhibited changes opposite to the changes in pro-inflammatory cytokines.3. Proportion and absolute number of Tregs in spleen CD4+T cells from C57BL/6, DBA/2 and BALB/c mice at different times after P.bANKA infectionThe percentage of Tregs in the spleen CD4+T cell population and the absolute number of Tregs increased after infection, with peaks on day 3 p.i. in C57BL/6, BALB/c, and DBA/2 mice respectively, and gradually declined from days 5-8 p.i.. In addition, the percentage and absolute number of Tregs in CM-susceptible C57BL/6 mice were significantly lower than in CM-resistant BALB/c and DBA/2 mice.4. Analysis of the effects of Tregs by in vivo CD25 depletionThe proportion of Tregs in the CD4+ T cell population derived from CD25-depleted C57BL/6, BALB/c and DBA/2 mice was lower than that of the control group. Tregs-depleted C57BL/6 mice had a significant increase in survival rate (80% survival on day 18 p.i.) and the parasitemia had a significant reductionon days 5,6, and 7 p.i. compared with intact mice. The clinical scores were also reduced in the CD25-depleted mice. The CD25-depleted mice did not develop ECM and survived until 3-4 weeks after infection when they developed hyperparasitemia and severe anemia. In contrast, CD25-depleted BALB/c mice had a similar course of parasitemia compared with intact mice, in addition to a temporary control of parasitemia during the early stage of acute infection with P.bANKA. Tregs enabled the rapid emergence on day 8 p.i. after CD25 depletion, thus preventing the enhanced T-cell activation controlling parasite growth with the resulting death of mice secondary to hyperparasitemia and severe anemia during weeks 3-4 after infection. CD25 depletion significantly reversed the production of IFN-γ, TNF-α, IL-6, IL-17, NO, and IL-10 on days 3 and 5 p.i. in the 3 mouse strains. CD25-depleted DBA/2 mice had a similar course of parasitemia and cytokine production compared with CD25-depleted BALB/c mice.5. The correlation analysis of Tregs with IFN-y, TNF-a, NO and IL-10 with parasitemia in C57BL/6, BALB/c and DBA/2 mice at different times after infection with P.bANKAThe frequency of Tregs during Plasmodium infection is negatively associated with the production of IFN-y, TNF-a, NO. However, the level of IL-10 during Plasmodium infection is positively associated with parasitemia.Conclusions1. Establishing an appropriate balance between pro-and anti-inflammatory immune responses is essential to control pathogenesis of severe malaria, and Tregs is an important regulator if this balance can be maintained.2. Tregs mediate the incidence and outcome of CM in P.bANKA infected mice by modifying the pro-inflammatory response to modulate the balance of pro-and anti-inflammatory responses.