Study On The Mechanism Of PKM2 In The Activation Of Myeloid Dendritic Cells From Patients With Severe Aplastic Anemia

Objective To explore the role PKM2 played in hyperfunction and hyperproliferation of myeloid dendritic cells(m DCs)and downstream effector T cells from patients with severe aplastic anemia(SAA),the level of PKM2 in m DCs from SAA patients and SAA mouse model was analyzed.The effect of different immunosuppressive agents on the function of PKM2 and m DCs was also investigated,which may provide a new way for studying the role of cellular metabolism in the immunopathogenesis of SAA,and provide the theoretical basis for targeted therapy of m DCs.Methods Untreated SAA patients,remission paitents,as well as normal controls were enrolled in this study.8 weeks old C57BL/6(Stock Number:213)and CB6F1(Stock Number:303)mice were obtained from Beijing Vital River laboratory animal technology corporation.Mice were housed in the Animal Center of Institute of Radiation Medicine Chinese Academy of Medical Sciences under SPF conditions.Part I The protein level of PKM2 in m DCs from 15 untreated SAA patients,15 remission SAA patients and 26 healthy controls were detected by flow cytometry and western blot,and the m RNA expression of PKM2 in m DCs were analyzed by Quantitative real-time PCR.Correlation among PKM2 levels,DC subsets and the blood routine was analyzed.Part II To observe the effect of PKM2 on the proliferation and activation of m DCs in patients with SAA,PKM2 in m DCs was knocked down using RNAi.The expression of CD80 and CD86 on m DCs were detected by flow cytometry,and the phagocytosis of m DC cells was detected by fluorescence immunospheres.Morphological changes of m DCs were observed under scanning electron microscope.Part ? To observe the effect of PKM2 on the activation of CD8 + T cells from SAA patients,m DCs were co-cultured with CTL(CD8 + T cells).The expression of perforin and granzyme m RNA in CTL cells were detected by real-time quantitative PCR.The levels of IFN-? in the supernatant were determined by ELISA.Apoptosis of CTL cells was detected by flow cytometry after staining with Annexin V.Part ? AA mouse model was established,and the proportion and functional moleculars of T subsets,m DCs(including CD80,CD86 and PKM2),Tregs were detected.The level of the expression of activating factor was also detected.After drug intervention with cyclosporine A and FK506,The above mentioned immune index were measured again.The glucose consumption,production level of pyruvate and ATP were also detected.Results Part I The level of PKM2 in the m DC cells in untreated group(59.1±15.8)% was significantly higher than those in remission group(42.6 ± 22.1)% and normal control(32.7 ± 20.2)%.The level of PKM2 was positively correlated with the proportion of DC cells,but negatively correlated with reticulocyte ratio,neutrophil absolute value and T subgroup ratio.The m RNA of PKM2 in m DC cells were(1.50±0.84)in untreated group,higher than those of remission group(0.81±0.24)and controls(0.32±0.11).The protein level of PKM2 in m DC cells in untreated group was also significantly increased.After immunosuppressive therapy,both the gene level and the protein level of PKM2 were gradually decreased.Part II After si RNA transfection,the views of the m DCs under SEM were round with typical morphology of short and little protrusions.The percentage of phagocytosis of m DC cells from control group(46.37%±7.6%)was higher than that of si PKM2 group(38.02%±4.4%)(p<0.05).The expression level of CD86 on m DC cells in si PKM2 group(41.89±5.54)% was significantly lower than that of control group(55.76 ± 7.08)%(P <0.05).Correlation analysis showed that PKM2 expression in m DCs from SAA patients was positively correlated with the expression level of CD86(= 0.562;=0.0126),but there was no significant correlation between the expression of PKM2 and CD80(=0.054;>0.05).Part ? The expression level of perforin m RNA in CD8 + T cells in si PKM2 group(0.84±0.39)was significantly lower than that in control group(1.23 ± 0.43).There was no significant difference of granzyme m RNA expression between si PKM2 group and the control group.The concentration of IFN-? in the culture supernatant in si PKM2 group(134.2 ± 25.1)was significantly lower than that in the control group(466.3 ± 53.9)by ELISA kit.Compared with the control group(34.86 ± 11.05)%,the apoptosis level of CTL cells in si PKM2 group was significantly increased(51.12 ± 14.96)%.Part ? In AA mouse model,the ratio of CD4+/CD8+ was decreased.The proportion of PKM2 and CD86 in m DCs was increased.The m RNA expression of perforin andgranzyme in CTLs was also elevated.The expression of Treg was decreased,and the level of PKM2 in m DC cells was significantly correlated with the function of m DC and T cells.After using cyclosporine and FK506 as immunosuppressive agents,the peripheral blood of AA mice was gradually recovered.The functional molecular levels of m DC cells and CD8+T cells were decreased,and the expression of PKM2 in m DC cells was significantly reduced.The levels of glucose consumption and production of pyruvate and ATP in m DC cells were lower than those before treatment(P <0.05).Conclusions 1.Comparing with remission group and normal control group,the level of PKM2 in the m DC cells in untreated group from SAA patients was significantly overexpressed both in the gene level and the protein level.The level of PKM2 in m DC was positively correlated with the level of DC cells,and negatively correlated with reticulocyte,ANC and T subgroup ratio.These results indicate that PKM2 may be involved in the activation of m DC cells in SAA patients and correlate with the degree of disease progression.2.After PKM2-si RNA transfection,the antigen uptake capacity of m DC was weakened.The expression of CD80 and CD86 on m DC from SAA patients was decreased,and the expression level of CD80 and CD86 was positively correlated with the level of PKM2 in m DC cells,which indicated that PKM2 in m DC cells from SAA patients could be an important factor resulting in the over-activation of m DCs.3.PKM2 can activate the function of downstream CTL cells and enhance the killing effect on target cells.4.The activation of PKM2 on m DC cells was confirmed in SAA mice.The level of PKM2 and the function of m DC cells was decreased after immunosuppressive therapy.PKM2 could be used as an index to evaluate the severity,efficacy and prognosis of SAA.This finding reveals the mechanism of action of cyclosporine and FK506 in the treatment of SAA,and provides a new idea for studying the role of cellular metabolism in SAA immunization.