Roles Of Tim-3 And The Phenotype Of Macrophages In Anti-infection Immunity

In recent years,infectious diseases caused by virus infection are increasing,which seriously endangers people's life and health.Recently,outbreaks of new and emerging viral infections such as influenza,Ebola and Zika have caused panic.In the meantime,some bioterrorism infections are extremely dangerous and the pathogens are complicated.It is known that the immune system plays a key role in defense against infection.How to understand the mechanism of the immune system to anti-infection response and seek new intervention strategies is an urgent scientific problem to be solved.In addition to the threat of individual pathogens,the combined exposure of multiple pathogens such as multiple bioterrorism agents poses a greater threat to human health.Although vaccines and other immune preparations are now available to respond to infections,how to formulate a reasonable and effective emergency immunization schedule,how to quickly and early evaluate the effect of combined immunization in the case of emergency co-immunization is another concern for researchers in the field.This dissertation mainly focuses on the mechanism and application of anti-infection immune response,and makes a preliminary exploration on the above two scientific issues.Tim-3(T-cell-Ig-mucin-3)is an immunosuppressive factor that was initially found to be expressed in activated Th1 cells,and its ligand Gal-9 binding can induce apoptosis and immune tolerance of T cells.Subsequent studies have found that Tim-3 also expressed in the activated Th17,Tc1 effector T cells,andits high expression can lead to decreased expression of IFN-?andIL-2 in effector cells,making cells in the state of disability or depletion(exchaustion)and anti-infective immune tolerance.In view of the relationship between the high expression of tim-3 and T-cell tolerance,the targeting of Tim-3antibody or Tim-3 fusion protein is considered to be a new strategy for T cell tolerance in infectious diseases such as HIV and HCV.Recently,our group found that Tim-3 also expressed in innate immune cells such as Macrophages,NK cells,and found that Tim-3plays an important role in maintaining the natural immune homeostasis,but by now,the mechanism by which Tim-3 inregulating innate immunity is not fully understood.Professor Kuchroo VKet al found Tim-3 may inhibit T cell function through inhibit Bat3 and CEACAM-1,but in the natural immune homeostasis,the molecular mechanism by which Tim-3regulatingremains to be fully understood.Our previous research found that Tim-3 can inhibit LPS/TLR mediated macrophage activation and promote immune tolerance.Then we found that Tim-3 can interact with STAT1,which in turn inhibits the latter's nuclear entry,resulting in imbalance of the immune homeostasis.It was also found that Tim-3 inhibits the phagocytosis of Listeria by macrophages by inhibiting the nuclear entry of Nrf2.Recently,the research group found that Tim-3 can inhibit the activation of the NOD-like receptor protein 3(NLRP3),which leads to immune imbalance.Based on the previous researches in innate immunity,the research group focuses deeper on innate immune by Tim-3 regulating,aims to further explain the potential molecular mechanisms of Tim-3 to regulate innate immunity.It is known that in the process of mammalian cell infection by virus,the genome or viral proteins induced by viral replication activate the innate immune signaling pathway and induce the production of interferon while inducing its downstream gene expression,promoting the anti-virus response of the cell;at the same time,the virus infecting host cells can also cause the accumulation of cytoplasmic particles,which are mainly composed of ribonucleoproteins,RNA-binding proteins,translation initiation factors,et al.This aggregation region is called stress granules(SG).Innate immune pathways and SG are both host cell responses to viral infections,whereas some viruses promote self-replication by inhibiting the formation of SG and by activing the innate immune pathways.Significantly,we found a new molecular-nuclear factor 90(NF90)involved in virus infection through the co-immunoprecipitation and mass spectrometry screening during the previous experiments.NF90 is encoded by the human interleukin-enhanced binding factor 3(ILF3)gene,and as a result of alternative splicing,it leads to the formation of multiple isoforms which make them a protein family.Studies have reported that NF90 play a key role in the biological growth and development,thus establishing the important position of this molecule;studies also found that NF90 can regulate a variety of cell cycle proteins,thereby regulating cell cycle progression,which further illustrates the important physiological role of NF90;Recently,many studies have reported the relationship between NF90 and viral replication and anti-virus infection.It has been reported that NF90 can promote the formation of SG by promoting the PKR/eIF2?phosphorylation pathway in virus infection,thereby inhibiting the initiation of the virus translation and finally exerting the immunological effect of anti-virus infection.There are two aspects of the current research focus on the NF90 molecule:The first is how the molecule affects the virus replication and subsequently antiviral response.The current thinking mechanisms are:1)NF90 inhibits viral specific cycles by binding to specific domains in viral RNA,reducing viral replication;2)NF90 can interact with viral core protein elements through the dsRBD domain,to antagonize the inhibitory effect of viral proteins on downstream signaling molecules;3)NF90 is an important component of stress granules(SG).NF90 inhibits the translation initiation of viral mRNA by promoting the formation of SG,and ultimately weakens viral replication.But there are still many unclear issues.The second is how NF90 itself is regulated,and there are many unknown in this aspect.This paper focuses on the second aspect of the research.In conclusion,NF90 plays an important role in the immune regulation of virus infection,and the previous study in innate immune regulation ofTim-3,We further explored the molecular mechanism of Tim-3 regulation to NF90 antiviral infection.VSV virus was used as the model of virus infection,successively infected steady knocking down Tim-3's RAW264.7 cell lines Tim-3-si and the peritoneal macrophage of whole knockout C57BL/6 Tim-3-KO mice with VSV,the corresponding wild-type macrophages were used as control.In the VSV infectious status:1)the NF90 gene and protein level were significantly increased not the knockdown but knockout Tim-3;2)VSV replication was inhibited;The above results suggest that Tim-3 may inhibit NF90-mediated infection tolerance by stimulating virus infection and at the same time proved that Tim-3 participates in and promotes virus infection.Then we conducted a further study on the molecular mechanismby which Tim-3 inhibits NF90 antiviral infection.Through experiments a new function-ubiquitination of NF90 was found.It was demonstrated that Tim-3 could promote the ubiquitination of NF90-Ub-K48 by overexpression Tim-3 in vitro and the peritoneal macrophages of Tim-3 transgenic mice.Furthermore,it was found that NF90-DZF-Domain was the domain of ubiquitination,and proved that Tim-3 could co-precipitation with DZF-Domain and promote its ubiquitination.Meanwhile,it is preliminarily confirmed that the intracellular segment of Tim-3-IC is a structural domain that plays important role of immune-negative regulation.Ubiquitination were found to be a new function of NF90 post-translation modification,the research group further screened the E3 ubiquitin ligase,which modified NF90's Ubiquitin,and through the gene chip and RT-PCR and immunoprecipitation technology we preliminary suspected TRIM16?TRIM47 and TRIM59are the E3 ubiquitin ligases which ptomoted NF90's Ubiquitin.In this part we found a new function of post-translationmodification-ubiquitinationof NF90 and through the experiment we proves that the concrete structure domain which Tim-3 regulates ubiquitination of NF90,but there are still many problems need to be further explored.Firstly,Tim-3 is a membrane protein,and NF90 is a nuclear protein,after the activation of the virus,it is a question of whether Tim-3 or NF90 is a scientific research question which is urgently needed to be answered by the research group?This problem will lay the foundation for the study of the exact molecular mechanism of Tim-3 against NF90.Secondly,The mechanism of the interaction between Tim-3 and NF90 and E3 ligase requires further examination.In addition,The ubiquitination-modified precise lysine sites in NF90-DZF-Domain becomes the next task for the research group to explore;Lastly,the precise amino acid residues and modification functions of the Tim-3-IC intracellular domain also need to be further explored and proved by the research team under the state of virus infection.In another part of our work,we explored a novel method for early assessment of combined immunological effects based on phenotypic changes of macrophages(M1 or M2 replication).Macrophages are known to be the first-line defense in the anti-infection immune response.Our previous results and literature studies found that macrophage polarization is closely related to the occurrence and development of diseases.For example,in the case of tumors,chronic infections and so on,macrophages are polarized toward the immunosuppressive M2 type,whereas in autoimmune diseases they are polarized toward the M1 type,which promotes inflammatory responses.In view of this,macrophages also become target cells for disease intervention.The results of our previous study confirmed that the polarization of macrophages is regulated by Tim-3molecule and its signal pathway is initially clarified,providing a new basis for the regulation of this cell.To further clarify the possible role of this cell in anti-infection immunity,we established a mouse model of the combination of plague and brucellosis and the analysis of the polarization and phenotype of mouse macrophages at early stage and the phenotype of T cells representing the ability of anti-infection response at late stage showed that at early stage of combined immunization,the phenotype of macrophages demonstrate consistent changes with the phenotype and function of CD8~+T cell,which showed anti-infection response at late stage,suggesting that detection of macrophage phenotype may be used to evaluate the effect of combined immunization early and rapidly.The results of this study provide a new way for us to rapidly evaluate the effect of co-immunization in emergent situations.