Novel Regulation Of Macrophage Polarization Based On Small Molecule

Diversity and plasticity are hallmarks of cells of the monocyte-macrophage lineage.As the essential components of the innate immune system,in addition to mediate inflammatory response and protect body against pathogens,macrophages also participated in organ remodeling and maintaining homeostasis.As a result,the specific macrophage function phenotypes play crucial but dissimilar roles at the inflammatory diseases,or the different stages of some certain disease.Because of this,exploring new regulatory mechanism of macrophage polarization and seeking for new key mediators will lay the foundation for the diagnosis and treatment of macrophage-related diseases.In order to further understand the regulatory net of macrophage polarization,we analyzed the difference of protein expression between M1 and M0 cells.Fortunately,we find a new mediator of M1 macrophage,the cytoplasm isoform of deoxyguanosine kinase.Our data shown that knockdown dGK significantly inhibited M1 macrophage associated pro-inflammatory factors in chapter 2.In addition,dGK knockdown also suppressed p-STAT1,whereas had no influence on p-NF-?B.So we hypothesized that STAT1 may be involved in the mechanism of dGK and designed assays to prove it by the utilizing of Jak activator(IFN-y)and inhibitor(Tofacitinib).Our observations suggested that dGK facilitated STAT1 activation induced by Jak2.In order to verify the role of dGK in vivo,we adoptive transferred dGK knockdown polarized-BMDMs to LPS challenged mice.And we found that dGK knockdown M1-BMDMs can not aggravate sepsis.All these data suggested that dGK increased Ml polarization through enhancing STAT1 activation.We also noted that dGK protein level was significantly reduced during the late M1 polarization,when cells suffered sever autophagy.Subsequent studies indicated the key role of autophagy in dGK degradation.And these findings also pointed out that autophagy was a negative regulator on the Ml macrophage.These observations completed dGK mechanism and provided explains for the regulation of autophagy on macrophage.Although the obviously modulation of dGK on macrophage polarization,it's hard to be the therapeutic targets due to the lack of significant difference between cytosolic and mitochondrial isoforms.So we turned our attention to a small compound,SND67,which had been studied previous,in order to find some novel targeting mechanisms.Based on this study,the effect of "molecular switch" mediated by TRAF6 on macrophage polarization was well detected.Firstly,we took advantage of small molecule compounds to investigate macrophage polarization.We have identified an activity compound,SND67,which induced the phenotype switch from M1 to M2 macrophage.In Chapter 3,we further studied its underlying mechanism.SND67 showed no effects on STAT1/STAT6 pathway and protein levels of IRF4 and IRF5.However,SND67 inhibited the translocated to nucleus of IRF5,while increased of IRF4.Further studies shown that this phenomenon was contributed to the disruption of IRF5 and TRAF6 interaction.Meanwhile,polyubiquitination level of IRF5 and IRF4 was also skewed.These observations indicated that the phenotype shift induced by SND67 was owned to the regulation on TRAF6-IRF5 and TRAF6-IRF4 interaction.Subsequently,collagen-induced arthritis and sepsis confirmed the anti-inflammatory effect of SND67 in vivo.SND67 significantly decreased the clinical scores and incidence rated of CIA mice.And joint swelling,bone damage and immune cells infiltration was also improved.Further,pro-inflammatory cytokines and II collagen antibody in serum was suppressed.These results indicate the amelioration of SND67 on rheumatoid arthritis mice.In addition,SND67 also improved LPS induced sepsis by increasing survival rate and decreased serum pro-inflammatory cytokines.Confocal assay confirmed the interaction of TRAF6 and IRF4 in peritoneal macrophages isolated from SND67 treated septic mice.In chapter 4 and 5,we further determined the influence of SND67 on TRAF6 and the relationship between TRAF6-IRF4 interaction and macrophage polarization.We proved TRAF6 was included in the targets of SND67 in vivo.And SPR was used to prove the direct interaction between SND67 and TRAF6.And the A and C ring of SND67 was considered as the activity group for its interaction with TRAF6.On the other hand,we analyzed the interaction between IRF4 and TRAF6 and found that the Ring domain of TRAF6 was required for the TRAF6-IRF4 interaction and the C70A mutant failed to induce K63-ubiquitination of IRF4 mediated by TRAF6.Consisted with this observations,the K63-ubiquitination of IRF4 was decreased when TRAF6 was knockdown,and the ubiquitination-IRF4 was mainly located in nucleus,which indicated that the ubiquitination was an activated modification of IRF4 and this activation could be mediated by TRAF6.Several cell-free systems were used to monitor their interaction and the results indicated a moderated affinity with the Kd value at 1?M.In summary,based on the macrophage polarization model,we identified two novel regulators.dGK promoted M1 polarization by enhancing STAT1 activation.But in the long-term polarized M1 cells,dGK was degraded by autophagy and the inflammatory response was calmed down slowly.On the other hand,owing to the unique activity of SND67,we discovered the effect of "molecular switch",TRAF6,on macrophage polarization.In the circumstance of M1 cells,SND67 induced the phenotype switch due to the turn off of TRAF6-IRF5 and turn on TRAF6-IRF4 interaction.All these observations further expended the regulatory net of macrophage polarization.The discovery of dGK and TRAF6 provided new potential therapeutics for inflammatory diseases,metabolic diseases and cancer.