The Study On Mechanism And Significance Of Tim-3 Regulating MHC-? Expression In Macrophages

Macrophages play an important role in the body's immune defense.It assist the recruitment and migration of immune cells by secreting chemokines and stimulating the production of adhesion molecules.Macrophages,as professional antigen-presenting cells widely exist in the body,participate in the initiation of specific immune response by activating T cell response through the expression of MHC-?/peptide complex.Antigen presentation mediated by MHC-? molecules plays an important role in transplant rejection,tumors and autoimmune diseases.The low expression of MHC-? on the surface of tumor cells can help them escape from the attack of specific immune cells.In autoimmune diseases,antigen-presenting cells can activate a large number of T cells by expressing MHC-? molecules,leading to an adaptive immune responses and causing autoimmune damage.However,the current regulation mechanism of MHC-? expression in macrophages remains to be elucidated.Multiple sclerosis is an inflammatory demyelinating disease of the nervous system with a high disability rate.With economic development and aging,the incidence rate is increasing year by year,so it has also received more and more attention.Multiple sclerosis is mainly caused by the overexpression of MHC-? molecules,which leads to abnormal activation of CD4⁺T cells and secretion of cytokines such as IFN-?and IL-17 to cause disease damage.These cytokines can also further regulate the secretion of inflammatory factors of macrophages,making the disease worse.Therefore,it has a profound significance to explore the antigen presentation function of macrophages.T cell immunoglobulin and mucin-domain containing-3(Tim-3)is a new star of immunosuppressive checkpoint molecule after PD-1 and CTLA4.Tim-3 is expressed on the surface of T cells,Tregs,DC,B cells,macrophages and NK cells.Abnormal immune tolerance induced by Tim-3 deletion is associated with asthma,allergies and autoimmune diseases.However,Tim-3 overexpression can inhibit T cell immune responses leading to immune exhaustion,which can induce chronic viral infection and cancer,and so on.Thus,the role of Tim-3 in diseases has received increasing attention and research.The activation of macrophages up-regulates the expression of Tim-3molecules on the surface.Under inflammatory stimulation,the expression of Tim-3 on the surface of macrophages is decreased and leads to the polarization of macrophages to the pro-inflammatory phenotype,secreting IL-1?,TNF?and IL-6,which can directly attack pathogens or induce the expression of cell surface adhesion molecules to promote cell migration.Tim-3 can also amplify immune responses by recruiting inflammatory cell infiltration through regulating the release of chemokines such as CCL9 and CCXL3 from macrophages.In addition,macrophage activation can also up-regulate the expression of intercellular adhesion molecules,helping immune cells to cross the immune barrier to reach the injured site to play a role.These above mechanisms not only participate in the immune defense process against bacterial and viral infections,but also play an important role in autoimmune diseases such as MS.Our previous research has shown that Tim-3 can affect the activation,polarization and phagocytosis of macrophages by regulating NF-?B,STAT1,Nrf2 and other signaling pathways,which are key molecules in the homeostatic regulation of macrophages Due to the important immune defense status of macrophages and the important role of Tim-3 for the regulation of their functions in defense against infection and autoimmune diseases,the research on the mechanism of Tim-3 regulating the function of macrophages has received great attention.MHC-? molecules are key to the establishment of immune tolerance to autoantigens.The aberrant recognition and presentation of autoantigens by MHC-? can lead to the occurrence of autoimmune diseases,such as MS and rheumatoid arthritis,among others.Tim-3,as an important immunosuppression checkpoint,is an important switch for immune cell activation.As the two key immune regulatory molecules of immune activity,whether there is an intrinsic connection between Tim-3 and MHC-? is currently unknown.Interestingly,we found that there is an inverse relationship between the expression of Tim-3 and MHC-? in human peripheral monocytes by analyzing the GEO database.So,can Tim-3 regulate the expression of MHC-? on macrophages?If so,what are the mechanisms by which Tim-3 regulates MHC-? and what are the implications in MS?It is desirable to intervene MHC-? mediated T cell activation through the exploration of these issues,providing new preventive and therapeutic approaches for MHC-?-related diseases.Objectives:1.Use the EAE model to explore the effect of Tim-3 on the expression of MHC-? on macrophages and its significance in MS;2.To explore the molecular mechanisms of Tim-3 regulating the expression of MHC-? on macrophages and the effect of Tim-3 on the phagocytic function of macrophages;Methods and results:1.Construction of EAE model and evaluation of Tim-3's influence on disease progressionWe first successfully constructed a MS model using Tim-3-TG,Tim-3KO,and WT mice.Comparison of clinical scores and body weight changes in Tim-3KO versus WT mice,Tim-3-TG versus WT mice,and mice injected with anti-Tim-3 antibody and control antibody,respectively,revealed more severe clinical signs and higher clinical scores in mice after Tim-3 knockdown or blockade.However,the clinical symptoms of Tim-3-TG mice were not as obvious as those of the WT group mice,and the scores were lower.Through tissue staining,blocking Tim-3 was found to aggravate inflammatory cell infiltration as well as loss of nerve fiber myelin in the mouse spinal cord.It shows that the down-regulation of Tim-3 is closely related to the progression of MS.Next,we explored the possible mechanisms by which Tim-3 affects MS2.In vivo intervention of Tim-3 function can affect the activation of CD4⁺T cells in MS by regulating the expression of MHC-? on the surface of macrophagesOn the basis of the negative association between Tim-3 and MHC-? found by GEO database analysis,we used WT and Tim-3-TG mice to construct EAE model,and detected the expression of MHC-? in peritoneal macrophages of EAE mice and the activation of splenic lymphocytes.It was found that after blocking the function of Tim-3,the gene and protein levels of MHC-? of peritoneal macrophages were significantly up-regulated,and IFN-?⁺CD4⁺T and IL-17⁺CD4⁺T cells in splenic lymphocytes were significantly increased.However,in the EAE constructed by Tim-3-TG mice,the expression of MHC-? was significantly lower than WT group,and IFN-?⁺CD4⁺T and IL-17⁺CD4⁺T cells in splenic lymphocytes obviously decrease in Tim-3-TG group.Then IAb-MOG_35-55-Tetramer was used to detect the specific TCL function on the surface of CD4⁺T cells,and the results showed that blocking Tim-3 resulted in increased activation of MHC-?/MOG_35-55-specific CD4⁺T cells.Due to the in vivo experiment's limitation,we examined the effect of Tim-3 on CD4⁺T by regulating the expression of MHC-? in macrophages by co-culturing peritoneal macrophages and splenocytes in vitro.we showed that the differentiation of IFN-?⁺CD4⁺T and IL-17⁺CD4⁺T cells induced by inhibition of Tim-3 were significantly reduced after neutralizing MHC-?.The above results indicate that Tim-3 may participate in the occurrence of multiple sclerosis by inhibiting the expression of MHC-?.In order to more clarify the regulatory effect and possible mechanism of Tim-3 on MHC-? in macrophages,we continued to explore it through in vitro experiments.3.Tim-3 inhibits the expression of MHC-? on macrophages in vitroRT-PCR,flow cytometry and Western Blotting were mainly used to detect the relationship between Tim-3 and MHC-? expression on RAW264.7 cells.The results showed that Tim-3 inhibited the expression of MHC-?.The same method was used to detect the expression of MHC-? on peritoneal macrophages of Tim-3-KO,Tim-3TG and WT mice,and WT treated with anti-Tim-3 antibody.The results all consistently showed that Tim-3 inhibited the expression of MHC-?.Subsequently,the Tim-3plasmid was transfected into HEK-293T cells through a gradient,and it was found that Tim-3 regulates the expression of MHC-? in a gradient-dependent manner.It was illustrated that Tim-3 inhibits the expression of MHC-? on macrophages.4.Tim-3 down-regulates MHC-? expression by inhibiting CIITATo further discuss the mechanisms of Tim-3 inhibiting the expression of MHC-?,we examined the effect of Tim-3 on CIITA,a transcriptional activator of MHC-?.RT-PCR and Western Blotting were used to detect the changes of CIITA expression in RAW264.7 cells after Tim-3 knockdown.The results showed that blocking Tim-3 could up-regulate the expression of CIITA.Next,the relationship between Tim-3 and CIITA was examined by knocking down CIITA in RAW264.7 cells.The results showed that after knocking down CIITA,Tim-3 had no significant regulatory effect on MHC-?.Subsequently,HEK-293T cells were transfected with CIITA and Tim-3 plasmids,and the expression of MHC-? was examined using Western blotting.The results similarly showed that Tim-3 down-regulated the expression of MHC-? by inhibiting CIITA.Finally,Tim-3 was used to interfere with the CIITA/MHC-? dual fluorescence reporter experiment,and the results indicated that Tim-3 inhibited the expression effect of CIITA on MHC-?.Together,it shows that Tim-3 down-regulates the expression of MHC-? on macrophages by inhibiting CIITA.5.Tim-3 down-regulates CIITA/MHC-? expression by inhibiting STAT1Through literature review,we found that STAT1 can regulate CIITA transcription,and previous experiments demonstrated that Tim-3 interacts with STAT1,so we examined the effect of Tim-3 on the expression of CIITA/MHC-? after inhibiting STAT1.First,STAT1 inhibitor was used to block the STAT1 signal of RAW264.7 cells,and the relationship between Tim-3 and MHC-? was detected by RT-PCR.The results showed that blocking STAT1 attenuated the enhancing effect of Tim-3 down-regulation on MHC-?.Then HEK-293T cells were transfected with STAT1 and CIITA,and the expression of CIITA and MHC-? was detected by Western Blotting.The results showed that STAT1 could promote the expression of both.Subsequently,HEK-293T cells were transfected with STAT1 and Tim-3,and the changes of CIITA and MHC-? were detected by RT-PCR and Western Blotting.It was found that Tim-3 blocked the up-regulation of CIITA and MHC-? by STAT1.Together,it shows that Tim-3 down-regulates the expression of MHC-? by inhibiting STAT1/CIITA.6.Tim-3 is involved in regulating macrophage phagocytosisThe phagocytosis of pathogens by macrophages is an important source of antigen presented by MHC-?.The preliminary experiments performed in the lab also proved that Tim-3 is able to inhibit the ability of macrophages to phagocytose bacteria.Therefore,we considered whether Tim-3 might affect the antigen presentation of MHC-? by inhibiting the phagocytic function of macrophages.We then explored the possible mechanisms by which Tim-3 regulates the phagocytic function of macrophages.The results showed that Tim-3 could mediate the phagocytic function of macrophages by inhibiting the Nrf2-COX-2 signaling pathway,thus intervening in the antigen presentation effect of macrophages,which in turn affected autoimmune homeostasis.Conclusions:1.Blocking the function of Tim-3 enhances the activation of CD4⁺T and aggravates the progress of EAE by up regulating the expression of MHC-? on macrophages.2.Tim-3 inhibits the expression of MHC-? on macrophages through STAT1/CIITA signaling pathway,and inhibits the phagocytosis of antigen by macrophages through COX-2.