The Role Of CD226/TIGIT-CD155 Signal In Allotransplantation By Regulating Macrophages Polarization

BackgroundComposite Tissue Allotransplantation(CTA)is an important or even the only effective therapeutic method for large-area tissue defect and dysfunction caused by severe trauma,congenital malformation,and tumor resection.Despite its positive effect on the repair of morphology and function,the patients have to take administration of immunosuppressant throughout their lives.Meanwhile,high-dose hormones and immunosuppressive agents often be applied to control alloimmunity when acute or chronic allograft rejection occurs.All these bring patients serious toxic side effects.Therefore,it is an urgent problem deserving enough attention and instant action to induce immune tolerance.As an important member of the natural immune response,Macrophages not only directly participate in the alloreaction towards allograft,but also act as antigen-presenting cells(APCs)which costimulate T cells and indirectly participate in alloimmunity.According to its difference in the phenotype and immune function,macrophages can be divided into M1 macrophages and M2 macrophages.M1 macrophages can secrete a series of pro-inflammatory cytokines,such as IL-1?,IL-6,IL-12 and IFN-?,which promote the occurrence of allotransplant rejection.However,M2 macrophages secrete antiinflammatory factors including IL-10 and TGF-?,and promote Treg cells proliferation and differentiation.So it involves in the induction of immune tolerance.Many studies have shown that CD226 and TIGIT expressing on T cells and NK cells serve as a co-stimulatory and co-inhibitory molecule respectively,which can competitively bind to CD155 expressing on APCs to activate or inhibit T cells and NK cells,respectively.However,the current studies have focused on the effects of CD226/TIGIT-CD155 signaling on the function of T cells and NK cells.Concerning its effect on APCs function,studies is rare.Recently,a research have reported that TIGIT can suppress CD4~+T cell activation and proliferation indirectly through the binding to CD155 on dendritic cells(DCs)to induce IL-10 section and suppressed IL-12 production.As a kind of APCs,CD155 also expressed on macrophages.Nevertheless,it is unknown when binding to CD155 whether CD226 and TIGIT play different roles in the process of regulating downstream signaling pathways to influence macrophage polarization and eventually participate in alloimmunity.Therefore,it is worthy to explore the role of CD226/TIGIT-CD155 signal in the regulation mechanism of macrophages polarization and their potential applications for induction of allograft immune tolerance.ObjectiveTo investigate the regulation effects and mechanism of CD226/TIGIT-CD155 signal on macrophages polarization,thus explore the scheme for inducing allograft tolerance.MethodsFirstly,peritoneal macrophages and spleen lymphocytes were co-cultured in a ratio of 1:5 in the presence of LPS and ?-CD3 mAb.The changes of CD155 expression on macrophages and CD226 and TIGIT expression on CD4~+ T cells were observed after coculture.Secondly,?-CD226 mAb or ?-TIGIT mAb was added to the co-culture system to block the CD226-CD155 signal and the TIGIT-CD155 signal respectively.Thus,it is feasible to observe the effect of intervening the CD226/TIGIT-CD155 signal on macrophages polarization and CD4~+T cells proliferation and differentiation.The M1-associated and M2-associated m RNA and protein level were detected to decide the polarization of macrophages.For example,q PCR were applied to detect the expression changes of m RNA,including M1 macrophages-associated genes such as IL-12p40,TNF-?,IL-1?,IL-6,i NOS,IFN-?,MCP-1 and M2 macrophages-associated genes such as Arg1,FIZZ1,YM1 and IL-10.And flow cytometry were applied to analyze changes of the ratio of CD11c~+CD206-M1 macrophages and CD11c-CD206~+ M2 macrophages.Besides,the proliferation of CD4~+T cells was detected by CFSE labeling of the lymphocytes based on the flow cytometry before and after co-culture respectively.And the changes of m RNA expression level of T-bet,GATA3,RORyt and Foxp3,which were the Th1?Th2?Th17 and Treg cells transcription factor respectively,were detected by q PCR.At the same time,the proportion of Treg cells was detected by flow cytometry.Due to the complexity of the co-culture system of macrophage and lymphocyte and the reciprocal interaction of multiple receptors-ligands,it will be convenient for us to use CD226-Fc and TIGIT-Fc on behalf of the CD226 and TIGIT signals provided by T cells to simulate the function of CD155 expressed on macrophages.And the results will be more accurate to reflect the effects of CD226 and TIGIT on CD155 expressed on macrophages.Macrophages were grouped as resting group(no any stimulation),Ctrl group(stimulated by LPS),CD226-Fc group(stimulated by LPS and CD226-Fc)and TIGIT-Fc group(stimulated by LPS and TIGIT-Fc).The effects of CD226-Fc and TIGIT-Fc on macrophage polarization were also examined by q PCR and flow cytometry to detect changes of m RNA and protein expression,as described above.Simultaneously,LUMINEX multi-factor assays were performed on cell culture supernatant to observe changes in the levels of IL-10,TNF-?,IL-12p70,MCP-1,IL-1?,IL-6,and IFN-?.Macrophages pretreated with CD226-Fc or TIGITFc were co-cultured with lymphocytes to observe the effect of macrophages pretreated with fusion protein on the proliferation and differentiation of CD4~+T cells.Finally,Western Blot was performed to examine the effects of CD226-Fc and TIGIT-Fc on the phosphorylation of SHP-1-ERK1/2-MSK1-CREB signal pathway which is closely related to the polarization of macrophages and secretion of IL-10.And meanwhile,nuclear translocation of IL-10 transcription factor CREB were investigated through immunofluorescence and Western Blot.In the in vivo experiments,we established a mouse model of skin allograft free transplantation using Balb/c mouse(H-2d)as a donor and C57BL/6 mouse(H-2b)as a recipient.The recipient mice were divided into three groups: WT group,CD226 KO group(CD226 molecule was knockout in the recipient mouse)and WT~+TIGIT-Fc group.Mice in all group received an intraperitoneal injection of 3 mg/kg rapamycin once a day at the first week,and once every other day from the second week to the fourth week post-transplanted.In the WT~+TIGIT-Fc group,recipient mice received 100?g of TIGIT-Fc on day 0 and day 7 after transplantation,while mice in the WT group and CD226 KO group received 100?g control antibody at the corresponding time.The effects of CD226 KO or TIGIT-Fc administration on the survival time of transplanted allografts were observed,and allografts in all groups were harvested at 4 weeks,6 weeks,and 8 weeks after transplantation to observe the pathological changes through HE staining.Moreover,the spleen and lymph nodes of the recipient mice were harvested at 4 weeks after transplantation to explore the effects of CD226 KO or TIGIT-Fc administration on the polarization of spleen macrophages and the percentage of Treg cells by flow cytometry.Finally,one-way mixed lymphocyte reaction was performed using the lymph node cells taken from the recipient mice as responder cells and MMC treated spleen lymphocytes derived from different strains of na?ve mice were used as stimulatory cells.Thus,the influences of CD226 KO or TIGIT-Fc administration on proliferation of recipient CD3~+T cells toward autoantigens,donor-specific antigens,and third-party antigens were detected.ResultsIn the co-culture system of macrophages and lymphocyte cells,the expression levels of CD155 on macrophages and CD226 and TIGIT on CD4~+ T cells were significantly increased after activated by co-culture.The addition of ?-CD226 mAb to block the CD226-CD155 signal in co-culture system significantly promoted the m RNA expression levels of M2 macrophages associated Arg1,FIZZ1 and IL-10,and inhibited the m RNA expression levels of M1 macrophages associated i NOS,IL-1? and IL-12p40.While addition of ?-TIGIT mAb to block the TIGIT-CD155 signal promoted the m RNA expression levels of M1 macrophages associated i NOS,IL-1? and TNF-?,and inhibited the m RNA expression levels of IL-10,which is a M2 macrophages associated gene.Besides,flow cytometry results showed that the addition of ?-CD226 mAb significantly promoted the polarization towards M2 macrophage and inhibited its polarization towards M1 macrophage.However,the addition of ?-TIGIT mAb plays an opposite regulatory role.Moreover,?-CD226 mAb could inhibit the proliferation of CD4~+T cells in the co-culture system,and promote the generation of Treg cells,and inhibit the m RNA expression levels of Th1 and Th17 transcription factors(T-bet and ROR?t respectively)and promote the m RNA expression level of Treg transcription factor(Foxp3).However,?-TIGIT mAb has an opposite effect on the proliferation and differentiation of CD4~+T cells.The results from q PCR,flow cytometry and LUMINEX have showed that,as the opposite function of CD226-Fc,TIGIT-Fc could promote the expression of M2 macrophages associated genes and inhibit the expression of M1 macrophages associated genes both from m RNA and protein levels.TIGIT-Fc could inhibit the phosphorylation of SHP-1 in macrophages,and promote the phosphorylation of ERK1/2-MSK1-CREB signal pathway,thus finally promote the nuclear translocation of IL-10 transcription factor CREB.However,CD226-Fc plays an opposite regulatory role.In addition,while macrophages pretreated with CD226-Fc promoted the differentiation of CD4~+ T cells to Th1,the macrophages pretreated by TIGIT-Fc inhibited the proliferation of CD4~+T cell and its differentiation to Th1 and Th17,and promoted differentiation to Treg.In the mouse model of skin allograft transplantation,CD226 KO or TIGIT-Fc administration significantly prolonged allograft survival time,and increased the proportion of Treg cells in the spleen and lymph nodes,and decreased the proportion of M1 macrophages and increased the proportion of M2 in the spleen.While there was no significant difference in the proliferation of recipient CD3~+T cells toward third-party antigens between all three groups,the proliferation of recipient CD3~+T cells toward donor antigens was significantly attenuated in mice from CD226 KO group and TIGIT-Fc administration group.In addition,the levels of pro-inflammatory cytokines in the serum of recipient mice,such as IL-12p70,TNF-?,IFN-?,MCP-1,IL-6,IL-1?,and IL-17 A,were significantly decreased,and the level of anti-inflammatory cytokine IL-10 was significantly increased.ConclusionCD226 and TIGIT competitively bind to CD155 expressing on macrophages and play an opposite role in regulating the downstream signaling pathway of CD155.TIGIT-CD155 signal promotes the transcription of IL-10 and the polarization towards M2 macrophages through ERK1/2-MSK1/CREB pathway.Inhibition of CD226 signal or promotion of TIGIT signal in vivo significantly prolongs skin allograft survival through promoting M2 macrophages polarization,Treg generation and IL-10 secretion,while inhibiting secretion of pro-inflammatory cytokines.Therefore,the CD226/TIGIT-CD155 signal could be a key molecular target to regulate alloimmunity.