Deficiency Of RNA Binding Protein QKI Promotes Tolerogenic Dendritic Cell Function Via IDO1-GCN2-eIF2α Signaling

Immune system is critical in the body in charge of providing immune response and maintaining the balance between immune activation and regulation.It is an efficient weapon against pathogens outside.Dendritic cell(DC)is the most important antigen presenting cell(APC),which functions on monitoring and regulating the immune system by highly efficiently capturing,processing and presenting antigens.Immature DCs have strong migration capability.When they turn to mature state,they gain the capacity to initiate and activate na?ve T cells,which puts them in the central position in immune response initiation,regulation and maintainance.Besides their ability in initiating immune response,DCs are also involved in the negative regulation of immunity.This two-way immunoregulation is highly associated with DCs’ fate determination and multiple genes involved in their function regulation[1,2].Tolerogenic dendritic cell belongs to a regulatory subset of DCs.It functions as a regulator in suppressing immunity and maintaining the immune balance.Tolerogenic DCs are characterized as low expression of co-stimulatory molecules and low migration capacity while possessing a high ability in secretion of immune regulatory chemokines such as TGF-β.It gains more and more attention recently with its potential in the application of autoimmune disease and organ transplantation.The metabolic state of cells is changed alongwith the environment.When subtle metabolic or environmental changes take place in the cells,integrated stress response(ISR)is induced,leading to activation of related transduction signals,thereby the synthesis and expression of proteins get suppressed[3].The expression and acitivation of many signal transduction genes is altered with the modification on different levels,including epigenetic,transcriptional,post transcriptional,translational,post translational,etc.Post transcriptional modification emerges as one of the most important regulation pathways.Through post transcriptional modification of signal transduction genes,the synthesis of specific protein get suppressed,followed by increased degradation.Therefore the metabolic state of the cells can be properly regulated to adjust to the environment changes[4].Regulation of metabolic state targeting DCs is possibly the reason and means to alter their way of immunoregulation[5].Amino acid metabolism is one of the most important members of cell metabolism.Indoleamine 2,3-dioxygenase 1(IDO1)is the first and rate-limiting enzyme of tryptophan catabolism through kynurenine pathway.Several reports have demonstrated that high expression of IDO1 is closely related with body immune inhibition and immune tolerance,by mediating multiple pathways on immunosuppression effects[6-12].RNA binding protein QKI belongs to STAR family,which functions as one of the most critical post-transcriptional factors taking part in the regulation of multiple organs and systems in the body.Previous reports have suggested that QKI plays a role in the differentiation and development of blood vessel and nervous system.According to our latest research,we showed that QKI also participates in the differentiation of colon epithelium as well as macrophages.However,the function and mechanism of QKI in DCs remain elucidated.In this thesis,we focus on the function of QKI on DCs and its specific mechanism in regulation.Objects: 1)To investigate the expression changes of QKI along with the differentiation and maturation of DCs.2)To discuss the effect of QKI knocking down on the proliferation and differentiation of DCs by an established Cre-ERTM-QKI-Loxp mouse model which enables specifically induced QKI knocking down in DCs.3)To detect the effect of QKI knocking down on the maturation,activation and secretion of DCs with the above model.4)To detect the effect of QKI knocking down in DCs on the activation of T cells and the differentiation fate determination of T cells.5)To investigate the function and specific mechanism of IDO1 metabolic pathway on DCs loss of QKI expression.Methods and Results: 1)First we successfully established the model of bone marrow-derived dendritic cell(BMDC)following the protocols.The model was verified by using RT-PCR,Western blot,FACS and ELISA to detect the markers of differentiation,maturation and activation of the induced DCs.Results from RT-PCR and Western blot showed that the expression of QKIis elevated along with the maturation process of BMDCs.This indicated that QKI may play a direct role in the maturation and regulation of DCs.2)We established the QKI specific knocking down BMDCs by the Cre-ERTM-QKI-Loxp mouse model.Results from FACS showed that the differentiation rate from mononuclear cell to DC is up-regulated after QKI knocking down.Meanwhile the proliferation level of DCs was raised.However,the surface expression of co-stimulatory molecules was significantly suppressed,which suggest that QKI knocking down probably suppress the immune activation of DCs.When QKI was overexpressed in DC by lenti virus vector,the suppressed expression of co-stimulatory molecules were partially recoverd.3)ELISA was applied to detect DC-related cytokines and chemokines using the specific QKI knocking down model above.The results exhibited that the expression of TGF-β was notablely up-regulated among all the cytokines detected in the knocking down BMDCs.Results from RT-PCR also showed that all the chemokines detected were suppressed in the QKI knocking down BMDCs,which would possibly restrains themigration of DCs.4)A co-culture of BMDCs and T cells seperated from splenic lymphocytes was carried on to observe the regulation of QKI in DCs on T cell activation and differentiation.QKI knocking down in BMDCs was shown to inhibit the capacity to express specific MHC I molecule,while promoting the expression of PD-L1 and PD-L2,both of which belong to the immunosuppressive ligands on immune cells.After co-culture of T cells and QKI knocking down BMDCs for 5 days,the differentiative capacity from CD4+T cells to Th1 cells was markedly restrained compared with co-culture with control BMDCs.On the contrary,the rate of differentiation into Treg was highly elevated.The secretion of immune activation cytokines,IL-2 and IFN-γ,were obviously down-regulated after T cells co-cultured with QKI knocking down BMDCs.In contrast,the secretion of immunosuppressive cytokine TGF-β was significantly up regulated.The results above indicated that DCs loss of QKI may change the differentiation fate determination of T cells through direct contact as well as paracrine secretion,leading to more Treg cells generation and less effector T cells or Th1 cells,which in turn promotes an immunosuppressvie environment.5)Afterwards,the specific mechanism of QKI on DCs was investigated.The ROS level of QKI knocking down BMDCs in steady state was shown to be obviouly lower than control group,detected by DCFH-DA probe.IDO1,known as an immunosuppression related ensyme,was found to be over expressed in the QKI knocking down BMDCs using RT-PCR and Western blot.The activation of IDO1-GCN2-e IF-2α pathway induced the ISR in DCs,resulting in elevation of autophagy and decreased protein synthesis.After treatment with 1-MT,one of the IDO1 specific inhibitors,the above pathway was inhibited to a certain degree.As a result,the immune response capacity in QKI knocking down BMDCs gained partial recovery.Conclusion: In this research,we for the first time discovered a strong corelation between specific knocking down the expression of RNA binding protein QKI and promotion to DC proliferation and differentiation.But at the same time,QKI knocking down exhibited aimmunosuppresive property by restraining the activation of expression of co-stimulatory molecules on DCs,simultaneously increasing the immunosuppressvie ligands’ expression.DC loss of QKI induced CD4+T cells differentiation into Treg cells through direct contact as well as paracrine scretion,causing an overall tolerogenic phenotype.Meanwhile we found that QKI was associated with the IDO1-GCN2 pathway involved in metabolic regulation.To conclude,the results above provide novel insights in the study of tolerogenic DCs,as well as the functional and mechanism investigation of the RNA binding protein QKI.Besides,the tolerogenic DCs generated in our study would potentially be applied in the clinical treatment of autoimmune disease and immunologic rejection after organ transplantation.