Study On The Effect Of MPLA Combined With R848 On Macrophage Polarization Into M1 Phenotype And Its Preliminary Mechanism

Macrophages are important immune cells with plasticity and function,which can polarize into M1 and M2 types under different environmental stimuli.M1 macrophage promotes the development of inflammation,mediates Th1 type immune response,and can eliminate invasive pathogens and tumor cells;M2 macrophage suppresses inflammatory reaction,mediates Th2 type immune response,and promotes the occurrence and development of tumors.TLR receptors recognize various pathogen-related molecular patterns,initiate intracellular signal transduction,activate immune cells,and play an important role in innate and adaptive immunity.Macrophages have a variety of TLR receptors,which can trigger their different polarization types and affect their biological functions after being activated.Our group is committed to the development of tumor vaccine adjuvants,and exploring the mechanism of TLR agonists combined application on macrophages is expected to provide theoretical support for the research and development of tumor vaccines.The purpose of this study was to explore the effect and mechanism of the combination of MPLA and R848 on the polarization of macrophages.Firstly,the effects of MPLA and R848 on the polarization and activation of macrophages were analyzed.Then,IL-4 was used to stimulate macrophages to induce M2 type macrophages,and whether MPLA combined with R848 could reverse M2 type macrophages into M1 type.Further,we analyzed the signal changes induced by MPLA combined with R848 in the induction of M1 macrophage polarization,and STAT3 inhibitor was used to analyze its effect on the combination of MPLA and R848induced-M1 macrophage polarization.The research is expected to provide a theoretical basis for the combined application of TLR agonists.The research mainly includes the following aspects:1.The combination of MPLA and R848 induces activation and the polarization of macrophages to M1 typeIn order to explore the effect of the combination of MPLA and R848 to induce activation and the polarization of macrophages,MPLA(500 ng/ml)and R848(2μg/ml)were used to stimulate RAW264.7 for 2h in vitro,and the effects of them on macrophages polarization and activation were detected by Griess reagent,q PCR and Western blot.The results showed that compared with the control group,the expression of i NOS and the secretion of NO were increased in MPLA or R848 treatment groups,while the expression of Arg-1 was decreased.The expression of i NOS and the secretion of NO were significantly increased after the combination treatment of the two agonists,while the expression of Arg-1 was significantly decreased,suggesting that the combination of the two agonists could synergistically induce macrophages to M1-type polarization.2.The combination of MPLA and R848 reverse M2 macrophages into M1 typeIn order to investigate whether MPLA and R848 could reverse M2 type macrophages into M1 type macrophages,IL-4(20 ng/ml)was used to stimulate mouse peritoneal macrophages to polarize them into M2 type macrophages.Then M2 type macrophages were co-cultured with MPLA(500 ng/ml)and R848(2μg/ml)for 2 hours,and the indexes of M1/M2 type macrophages were detected by q PCR.The results showed that the expression of i NOS was slightly higher than the control group,and the expression of Arg-1 was slightly lower than IL-4 group;The expression of i NOS in the combination group of two agonists was significantly higher than that in the control group and other experimental groups,and the expression of Arg-1 was significantly lower than that in other groups,suggesting that the combination of MPLA and R848 could reverse M2 type macrophages into M1 type macrophages.3.The effect of the combination of MPLA and R848 on AKT,STAT3,STAT1,SOCS3,SOCS1 and IκBNext,we explore the mechanism of the combination of MPLA and R848 to induce macrophages to polarize to M1 type.After using MPLA(500 ng/ml)and R848(2μg/ml)alone or in combination to stimulate RAW264.7 2h,western blot and q PCR were used to analyze the expression of AKT,STAT3,STAT1,SOCS3,SOCS1 and IκB.The results showed that the expression of p-STAT3/STAT3,SOCS3,p-IκB/IκB molecules showed an regulating trend compared with the control group after MPLA and R848 agonists treated alone.The increase effect was most significant after the combination of the two drugs;compared with the control group,the expression of p-AKT/AKT,p-STAT1/STAT1 and SOCS1 molecules showed a downward trend,with the most significant effect after the combination of the two drugs.4.After adding STAT3 inhibitor,the effect of STAT3 on M1-type polarization of macrophages in the combined application of MPLA and R848 was analyzedThis study found that the change of STAT3 molecule after stimulation with MPLA and R848 was consistent with the change trend of i NOS.Therefore,in order to verify the role of STAT3 in macrophage polarization,STAT3 inhibitor(10μM/ml)were added,and used western blot and q PCR to detect the change of i NOS,Arg-1 and related pathway signals.The results showed that after adding STAT3 inhibitor,the expression of i NOS was lower than that of the agonist alone treatment group,suggesting that the synergistic effect of MPLA and R848 on inducing macrophages to polarize to M1 type was relieved.There was no significant difference in the expression of Arg-1.At the same time,the change trend of SOCS3 and STAT3 were both decreased compared with the agonist alone treatment group.The changes of STAT1 and SOCS1 were not significant.Conclusion: This study shows that the combination of MPLA and R848 can synergistically induce M1 type polarization of macrophages,and can reverse the M2 type macrophages induced by IL-4 to M1 type.In this process,STAT3 plays an important role in the M1 type polarization induced by MPLA and R848.The study may provide a theoretical basis for the cross-talk mechanism of TLRs in immune cells and the subsequent research and development of tumor vaccine adjuvants.