| Macrophages are highly heterogeneous and plastic.They can differentiate into two types of polarization:M1?Classically activated macrophage,M1?and M2?Alternatively activated macrophage,M2?under different microenvironmental stimulation.Phages can also drift and become the opposite functional phenotype[1].M1participates in the acute pro-inflammatory response to promote Th1-type immunity,to clear invading pathogens and tumor cells;while M2 suppresses the inflammatory response to promote Th2-type immunity to help tumor growth.Toll-like receptor is a natural pattern recognition receptor widely expressed in various tissues and organs of an organism,which playing an important role in innate and adaptive immunity.The research on the combination of TLR agonists have become one of the hot issues in the field of tumor-immunology.TLRs ligands have been used as immunological adjuvants in tumor immunotherapy.These TLR ligands can be used alone,or they can be used in combination to further enhance the immune effect of the vaccine.Our research group is dedicated to the development of tumor immune adjuvants,and to explore the role of TLRs ligands in the regulation of M2 macrophage phenotypic transformation,which will provide new strategies for tumor immunotherapy.The purpose of this study was to investigate the effects of Pam3CSK4,LPS and R848 on the polarization of macrophages and their regulatory effects.The study first used in vitro experiments to analyze the direct effect of Pam3CSK4,LPS and R848 on the typing of macrophages.Further,the M2 type macrophage model was established by the tumor culture supernatant to explore the combination of Pam3CSK4,LPS and R848 on M2—regulatory role of M1 macrophage turnover.The research can provide a theoretical basis for the further application of TLRs agonists.The research contents of this study include the following aspects:1.The combination of Pam3CSK4,LPS and R848 induce macrophage activation and polarizing to M1 type1.1 Pam3CSK4,LPS and R848 combine to change the morphology of macrophagesThe peritoneal macrophages of mice were stimulated with Pam3CSK4,LPS and R848 separately or in combination for 24 hours.Microscopic observation of macrophages showed obvious morphological changes.It is manifested that when macrophages are stimulated with Pam3CSK4,LPS and R848 alone,compared with the control group,the foamy morphology of each group has increased;when Pam3CSK4,LPS and R848 are used in combination,their morphology changes was more significant than that of the group used alone.These results suggesting that they may have the best stimulation effect.1.2 Effect of Pam3CSK4,LPS and R848 combination on macrophage activationTo investigate the activation of Pam3CSK4,LPS and R848 on macrophages,Pam3CSK4,LPS and R848 alone or in combination stimulated mouse peritoneal macrophages,mouse bone marrow-derived macrophages and RAW264.7 cells for 24hours.The secretion of NO,IL-1?,TNF-?and IL-6 of activating macrophage were detected by using Griess reagent and ELISA method.The results showed similar trends in all three types of macrophages:when Pam3CSK4,LPS,and R848 were used alone,R848 promoted macrophages to secrete NO,IL-1?,TNF-?,and IL-6 than the other two agonists;when Pam3CSK4,LPS and R848 are used in combination,compared with the other two groups,LPS and R848 can promote the macrophages to secrete NO,IL-1?,TNF-?and IL-6.Pam3CSK4 Combined with R848 can weaken the ability of R848 which promote the secretion of NO,IL-1?,TNF-?and IL-6 by macrophages;combined with Pam3CSK4,LPS and R848,compared with all groups,it can promote synergistic promotion of macrophage NO,IL-1?,TNF-?and IL-6 secretion?P<0.05?.These results suggested that the combination of Pam3CSK4,LPS and R848 can activate macrophages.1.3 Effect of Pam3CSK4,LPS and R848 combination on macrophage polarizationFurther,the effects of Pam3CSK4,LPS and R848 on macrophage polarization were explored.Pam3CSK4,LPS and R848 alone or in combination stimulated mouse peritoneal macrophages,mouse bone marrow-derived macrophages,and RAW264.7for 24 hours.qPCR combined with ELISA was used to detect the expression of iNOS,IL-12 and M2 macrophages.The results showed that among the three macrophages,when Pam3CSK4,LPS and R848 were used alone,R848 up-regulated the expression of iNOS and IL-12 of macrophages.Compared with the control group,the combination of LPS and R848 in promoting macrophages to express iNOS and IL-12 is better,while the combination of Pam3CSK4 and R848 can weaken the ability of R848 which promote iNOS and IL-12 in macrophages;the combination of Pam3CSK4,LPS and R848 to promote iNOS and IL-12 in macrophages was the strongest?P<0.05?.In contrast,Pam3CSK4,LPS and R848 alone or in combination stimulated macrophages,all of which inhibited the expression of Arg-1,IL-10,and Pam3CSK4,LPS,and R848had the strongest inhibitory effect.2.The combination of Pam3CSK4,LPS and R848 reverse M2 macrophages into M1 typeThis paper explores the role of Pam3CSK4,LPS,and R848 in regulating M2macrophages into M1 type.First,Lewis lung cancer tumor cell culture supernatant was usedthe to mimic the tumor microenvironment to induce mouse peritoneal macrophages into M2 macrophage model.Griess reagent,qPCR and ELISA methods were used to detect the markers of M1 and M2 macrophage polarization,which verify the success of the model.TAM?M2 macrophages?was then cultured alone or in combination with Pam3CSK4,LPS and R848 for 48 h.The above experimental method was used to detect the whether the combination could reverse the M2 into M1.The results showed that Pam3CSK4,LPS and R848 alone could not reverse M2macrophages into M1.Pam3CSK4 combined with LPS or R848,LPS combined with R848 can reverse M2 macrophages to M1 flip to a certain degree.When Pam3CSK4,LPS,and R848 are combined,the flipping effect is the strongest.3.Preliminary study on the mechanism of the combination of Pam3CSK4,LPS and R848 inducing macrophages to polarize to M1In this study,the mechanism of Pam3CSK4,LPS and R848 inducing macrophages to polarize to M1 was explored.First,qPCR method was used to detect the expression of TRAF6 and TRAF3 mRNA on macrophages in each group.The results showed that among the Pam3CSK4,LPS,and R848 alone groups,the TRAF6expression was highest in the R848 group;in the Pam3CSK4,LPS,and R848two-in-one combination group,the LPS and R848 combined group had the highest TRAF6 expression;when Pam3CSK4,LPS,and R848 were combined,Compared with all the groups,the expression of TRAF6 was the highest on macrophages,suggesting that the expression of TRAF6 is highly correlated with the degree of polarization of M1 macrophages.The analysis of TRAF3 mRNA expression showed that the levels of TRAF3 in macrophages treated with TLRs agonists were lower than those in the control group,and the expression of TRAF3 mRNA was lowest in the combined treatment group of Pam3CSK4,LPS and R848.The above results indicate that the promotion of M1 polarization of macrophages by Pam3CSK4,LPS and R848 may be related to the up-regulation of TRAF6 expression and the down-regulation of TRAF3expression.Therefore,the study then investigated the upstream signal of TRIF.The results showed that LPS can promote the expression of TRIF in macrophages;the combination of Pam3CSK4 and LPS,the combination of LPS and R848 can synergistically promote the expression of TRIF in macrophages,and the combined promotion effect of LPS and R848 is stronger.When Pam3CSK4,LPS,and R848 were combined,TRIF expression on macrophages was shown to be up-regulated and higher than other groups.It is suggested that the promotion of M1 polarization of macrophages by Pam3CSK4,LPS and R848 may be related to the balance of TRAF6/3 mediated by TRIF.Based on existing research reports,we believe that in the TLRs signaling pathway,TRAF6 can promote macrophage activation and polarize to M1,while TRAF3 plays the opposite role.The specific mechanism of TRAF3 and TRAF6regulating macrophage M1 polarization is under further study.In summary,this study shows that Pam3CSK4,LPS,and R848 can directly induce macrophages to polarize to M1 type,and can reverse TCS-induced M2macrophages to M1 type.It is suggested that Pam3CSK4,LPS and R848 may play an anti-tumor role by regulating the polarization type of macrophages,and lay an experimental foundation for the further development of effective tumor treatment and even tumor prevention biological drugs. |
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