Roles And Regulations Of Glycogen Metabolism In Macrophage Polarization And Memory T Cell Maintenance

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Role of glycogen metabolism in the polarization and survival of M1 macrophageObjective:To explore whether glycogen metabolism is involved in M1 macrophages polarization,we study the influence of glycogen metabolism on M1 macrophages differentiation,function and survival.Furthemore,we also investigate the affect of pentose phosphate pathway(PPP)on M1 macrophages polarization..Methods:(1)Glycogen levels in bone marrow-derived M0,M1 and M2 macrophages were detected by TEM,PAS staining,colorimetric assay,the expression of enzymes involved in glycogen metabolism was analyzed by Real-time PCR.(2)The glucose consumption rate was measured by enzymatic methods.The expression of genes involved in transport of glucose and hexokinase was analyzed by Real-time PCR.(3)Ml macrophages were transfccted with Gysl and Pygl siRNAs,and the expression of Ml macrophages related genes were analyzed by Real-time PCR.macrophages related genes was analyzed by Real-time PCR,the cytokines and NO levels were furthcer analyzed by ELISA and NO assay kit respectively.(4)Gysl and Pygl siRNAs were transfected into M1 macrophages,and the release of LDH from M1 macrophages was detected.(5)M1 macrophages were treated with GPI,and the ROS levels were detected by CellROX(?)green flow cytometry assay.(6)The expression of genes involved in pentose phosphate pathway was analyzed in MO,M1 and M2 macrophages by Real-time PCR,the NADPH/NADP and GSH/GSSG ratio was analyzed by colorimetric assay and fluorometric assay.(7)M1 macrophages were treated with GPI,6AN or OT,and the NADPH/NADP ratio and glycogen levels were measured by colorimetric assay.Meanwhile,the GSH/GSSG ratio was detected by fluorometric assay.And M1 macrophages were treated with GPI,6AN or OT with or without GSH,the release of LDH from M1 macrophages was detected.(8)M1 macrophages were treated with 6AN or OT,the expression of M1 macrophages related genes were analyzed by Real-time PCR,the cytokines and NO level were analyzed by ELISA and NO assay kit.(9)M1 macrophages were treated with G6PDH inhibitor,6AN for 6 hours,the expression of Ml macrophages related genes were analyzed by Real-time PCR.Meanwhile,the cytokines and NO level were analyzed by ELISA and NO assay kit.(10)The serum from the LPS-induced septic shock or conA-induced hepatitis mice treated with GPI was collected,the cytokines,NO level and ALT level were detected by ELISA,NO assay kit and ALT assay kit,respectively.Meanwhile,the survival of mice were monitored.Results:(1)M1 nmacrophages had much higher glycogen levels,compared to MO or M2 macrophage.And the expression of genes involved in glycogen metabolism was upregulated in M1 macrophages.(2)Compared to MO and M2 macrophages,the glucose consumption rate or the expression of hexokinase or genes involved in transport of glucose were increased.(3)Inhibition of Gysl ancd Pygl enzymatic activity affected the polarization of M1 nmacrophages.(4)Inhibition of glycogen levels by Gys1 siRNA or Pyg1 siRNA or GPI in M1 macropahges could increase the release of LDH.(5)Inhibition of glycogen degrade by GPI in M1 macrophages could increase the ROS levels.(6)The pentose phosphate pathway and the antioxidant activity of M1 macrophages were upregulated compared with MO macrophages.(7)Exogenous GSH could rescue the macrophage damage induced by the suppression of glycogen-PPP metabolic pathway.(8)Inhibition of PPP metabolism affacted the polarization,function and survival of M1 macrophages.(9)Inhibition of NOX2 enzymatic activity could affect the polarization and function of M1 macrophages.(10)GPI treatment could attenuate the secretion of serum cytokines in LPS-challenged or conA-induced mice.Conclusions:The expression of hexokinase and the enzymes involved in transport of glucose was upreglulated in Ml macrophages and the resultant G6P can be channeled to glycogen,and the stock pile of glycogen is utilized to maintain the supplies of G6P.In coupling with the pentose phosphate pathway,vast G6P is oxidized to generate NADPH to ensure high levels of reduced glutathione,which is essential for quenching ROS so to facilitate the polarization and function of M1 macrophages.However,lower ROS level also influences the polarization and function of M1 macrophages,Elucidation of glycogen-PPP metabolism pathway in Ml macrophages polarization and survival provides a potential target for inflammation treatment.Part ? PCK1-directed glycogen metabolic program regulates memory CD8+ T cell formation and maintenanceObjective:Metabolic activities play a crucial role in controlling memory T cell homeostasis.To study the mechanisms linking metabolic signals to memory cell formation and survival.In vivo OVA-specific CD8+Tm cells and in vitro IL-15 derived CD8+Tm were generated using adoptive transfer model or IL-15 treatment.Then we investigate the effect of gluconeogenesis,glycogen metabolism and the pentose phosphate pathway during CD8+ Tm cell formation and homeostasis.Methods:(1)CD8+ Tm cells were sorted from mice transferred with OT-1 CD8+T cells or induced by IL-15 in vitro,the expression of PCK1,FBP1 and G6pase in gluconeogenic were determined by Real-time PCR and western blot.(2)Glycogen levels in CD8+ Tm cells were detected by electronic microscope and colorimetric assay,and the experession of genes involved in glycogen metabolism was analyzed by Real-time PCR.(3)Glycogen concentration in IL-15 CD8+Tm treated with 3-MPA and PCK1+/-OT-I derived CD8+Tm cells were measured by colorimetric assay.(4)Lm-OVA-induced CD8+ Tm cells were stimulated with OVA257-26a in the presence of GPI,the marker of effect T cell were measured by ELISA and FCM.(5)C57BL/6 mice,adoptively transferred with CD45.1+OT-I CD8+T cells,were infected with Lm-OVA,meanwhile mice were intraperitoneal injected with GPI and control saline.On day 6 and day 30,the CD45.1+CD8+T cells were analyzed by FCM.(6)We adoptively transferred wild-type or PCK1+/-OT-I T cells into CD45.1 mice and followed by infection with Lm-OVA.30 days later,mice were sacrificed and the CD45.2+CD8+T cells in spleen,lymph nodes and peripheral blood were analyzed by FCM.(7)IL-15 CD8+Tm cells were treated with GPI,6AN or 3-MPA,and the NADPH/NADP ratio and glycogen levels were analyzed by colorimetric assay.(8)IL-15 CD8+Tm cells were treated with GPI;6AN,BSO or DNCB,the GSII/GSSG ratio,the ROS level and IL-15 number were detected by fluorometric assay or FCM.(9)WT mice were adoptively transferred with CD45.1+ OT-I CD8+ T cells followed by Lm-OVA infection.30 days later,mice were treated with 3-MPA,GPI or 6AN with or without GSH,30 days later the ClD45.1?CD8+T cells were analyzed by FCM.(10)WT mice were adoptively transferred with CD45.1+OT-I CD8+T cells then inf-ected Lm-OVA,meanwhile mice were administrated with 3-MPA or GPI or control saline.On day 30,the above mice were inoculated subcutaneously with B 16-OVA cells.The growth of tumor and the survival of mice were monitored.And the PCK+/-mice and PCK1 over expression CD8+ Tm were used to analyze the growth of tumor.Results:(1)The expression of PCK1 and FBP1 were upregulated in CD8+ Tm cells both in vivo and in vitro.(2)The glycogen level and the genes involved in glycogen metabolism of Tm were upregulated compared with Tn and Teff.(3)Inhibition of PCK1 decreased glycogen level in IL-15-induced CD8+Tm cells.(4)GPI treatment did not affect the recall response of CD8+ Tm.(5)GPI inhibits CD8+Tm cells formation in vivo.(6)PCK1 knockdown decreases CD8+Tm cell formation in vivo.(7)PCK1-Glycogen-PPP pathway is required for CD8+ Tm cell survival.(8)CD8+ Tm cells effectively use the glutathione system to clear ROS for their survival.(9)GSH rescued impairment of ROS on memory T cell survival.(10)The mice treated with 3-MPA or GPI showed accelerated tumor growth.However,PCK1-overexpressing OT-1 CD8+ T cells resulted in improved antitumor effect against B 16-OVA melanoma in mice.Conclusions:CD8+ Tm cells,by virtue of their overt upregulating gluconeogenic enzyme PCKI and enzymes for glycogen metabolism,boost the biosynthesis of G6P along the gluconeogenic pathway.As a result,abundant G6P can be channeled to glycogen,and the stock pile of glycogen is utilized to maintain the supplies of G6P.In coupling with the pentose phosphate pathway,vast G6P is oxidized to generate NADPH to ensure high levels of reduced glutathione,which is essential for quenching ROS so to facilitate the survival and fate maintenance of CD8+ Tm cells.Revelation of this molecular mechanism of CD8+ Tm formation and maintenance might bring new approach to regulate T cell memory with great potential of clinical applications.