| Solid organ transplantation is a treatment strategy that can treat many end-stage organ failures.For example,liver transplantation is the best option for the treatment of end-stage liver diseases such as liver cancer and liver failure.The rejection that occurs in the body after transplantation remains a major constraint on the efficacy of transplantation.Survival after solid organ transplantation is limited by chronic rejection and the need for lifelong use of immunosuppression and its associated toxicity.For example,after liver transplantation,the recipient will always experience a more or less severe immune response and a series of complications that pose a certain threat to the patient’s health.The acute rejection of organ transplantation is an important factor affecting the survival of patients after organ transplantation,and is a hot spot and a difficult area of research at present.The mechanism underlying acute rejection has yet to be further elucidated.In order to ameliorate the phenomenon of rejection after patients undergo organ transplantation,many immunosuppressive drugs have appeared in the market,which need to be taken for a long time or even for life,and such a situation will bring a great burden to patients.Therefore,several preclinical and clinical studies have begun to explore ways to induce immune tolerance to transplantation without the need for lifelong immunosuppression.Dendritic cells(DCs)are specialized antigen-presenting cells(APCs)known for their ability to initiate the immune system,but some studies have shown that they can promote tolerance to allogeneic antigens.Both myeloid and plasmacytoid dendritic cells(pDCs)can be involved in tolerance to allogeneic antigens.It has even been shown that the function of DC cells is correlated with their maturity,with mature DCs being able to activate the immune response,but immature DCs being more likely to induce tolerance to allogens.Therefore,maintaining the immature state of DCs in vivo is crucial for inducing tolerance function.Previous studies have shown that many miRNAs are abnormally expressed during DC maturation,and miR-let-7i is upregulated during this process.Previous studies on the effects of miR-let-7i on DC cells were mostly in vitro studies and tumor immunization studies,but in vivo studies on the effects of miR-let-7i on DC maturation and function and its induction of immune tolerance in animal allografts are innovative.In previous studies,the maintenance of DC immaturity has been shown to be effective in rodent models,but it is difficult to induce the same effect in primate models.Therefore,this study was conducted in rhesus monkeys,whose immune system is highly similar to that of humans.The induction of targeted and durable immune tolerance in non-human primates will have important theoretical implications and clinical applications.In this study,we used non-human primates,rhesus monkeys,as test animals to construct a rhesus monkey allograft model to investigate the role and mechanism of inhibition of DC cell miR-let-7i in the induction of skin immune tolerance in rhesus monkeys,which is theoretically valuable and innovative.Objective:1.Dendritic cells(DC)in peripheral blood of rhesus monkeys were isolated,induced and identified;DC cells were mixed with T lymphocytes and cultured to investigate the functions of DC cells in different states and their effects on T cells.2.To explore the role of miR-let-7i in the immature state and function of DCs.3.To investigate the molecular mechanism by which miR-let-7i promotes DC maintenance of immature state and immune tolerance function.4.To establish an animal model to explore the effect of miR-let-7i on immune tolerance of skin grafts in rhesus monkeys.Methods:1.isolation of rhesus peripheral blood and collection of CD34+cells using mononuclear cell isolate.2.induction of immature DC(imDC)cells using granulocyte-macrophage colony factor(GM-CSF),interleukin(IL)-4;induction of mature DC(mDC)by TNF-α.3.microscopic observation of cell morphology.4.flow cytometry for phenotypic identification of cells and detection of DC maturation marker expression.5.CCK-8 assay for proliferation of mixed culture T lymphocytes.6.flow cytometry to detect T-cell apoptosis.7.enzyme-linked immunosorbent assay(ELISA)for the detection of IL-2,IL-4,IL-10 and IFN-y expression in the supernatant.8.construction of overexpression/low expression miRNAlet-7i vectors(synthetic mimic/inhibitor liposome transfection)and parallel transfection efficiency assay.9.real-time fluorescence quantitative PCR(RT-qPCR)to detect miR-let-7i expression.10.phenotypic identification of cells after gene transfection by flow cytometry.11.CCK-8 assay for T cell proliferation after gene transfection.12.flow cytometry detection of T cell apoptosis after gene transfection.13.ELISA to detect IL-2,IL-4,IL-10 and IFN-γ expression in the supernatant after gene transfection.14.flow cytometry to detect the proportion of CD4+CD25+Treg cells after gene transfection.15.bioinformatics website(StarBase)to predict miR-let-7i binding sites to CCR7.16.dual luciferase reporter gene validation of miR-let-7i in relation to CCR7 targeting.17.protein blotting(Western Blot)to verify the regulatory relationship between miR-let-7i and CCR7 and related protein expression.18.CCK-8 assay of the effect of miRNAlet-7i and CCR7 on the proliferation viability of T cells under different treatment factors.19.flow cytometry to detect the effect of miRNAlet-7i and CCR7 on T cell apoptosis under different treatment factors.20.flow cytometry to detect the effect of miRNAlet-7i and CCR7 on CD4+CD25+Treg cell differentiation under different treatment factors.21.ELISA to detect the effect of miRNAlet-7i and CCR7 on the expression of inflammatory factors IL-2,IL-4,IL-10 and IFN-y in cells with different treatment factors.22.construction of a rhesus monkey allogeneic skin graft model.23.A study to investigate the effect of inhibiting miRNAlet-7i signaling in dendritic cells on skin slice rejection after allografting in rhesus monkeys.Results:1.The isolated CD34+cells were successfully induced with imDC on day 5,and the cell surface was smooth and uneven with few burr protrusions observed under the microscope.mDC were successfully induced by culture until day 7,and there were many dendritic protrusions on the cell surface with different lengths and morphologies observed under the microscope.2.miR-let-7i expression was found to be elevated during TNF-α-induced DC maturation by real-time fluorescence quantitative PCR(P<0.01);miR-let-7i mimic was transfected with miR-let-7i inhibitor into cells,and miR-let-7i expression was elevated versus significantly decreased.It shows that the transfection is successful,the transfection effect is good,and the reagents used in this experiment can effectively regulate the expression level of miRNA-let-7i.3.flow detection of DC surface maturation marker expression,after DC induction using LPS,CD80,CD86,MHC Ⅱ positive rates were 25.1%,20.6%,36.8%,which were significantly higher compared to imDC cells.While transfecting miR-let-7i mimic into cells its effect of promoting LPS induction,CD80,CD86,MHC Ⅱ positive rates of 97.6%,91.9%,96.6%were significantly different compared to NC-mimic+LPS group,but overexpression of miR-let-7i was accompanied by simultaneous overexpression of CCR7,miR-let-7i overexpression was significantly reversed,while after suppressing miR-let-7i expression,the positive rates of CD80,CD86,and MHC Ⅱ were:40.9%,41.3%,and 41.7%,and the LPS-induced effect was significantly reversed and significantly different,after reducing miR-let-7i while also reducing CCR7 expression,the positive rates of CD80,CD86,and MHC Ⅱ positivity rates were again increased.Overexpression of CCR7 alone after LPS induction significantly inhibited the effect of LPS induction,while the addition of Toll-like receptor signaling pathway agonist partially reversed the effect of overexpression of CCR7;reduction of CCR7 expression significantly promoted the effect of LPS induction,but the addition of Toll-like receptor pathway inhibitor partially reversed the effect of low expression of CCR7.4.CCK-8 assay found that mDC significantly promoted the proliferation viability of T cells,and when the ratio of the two reached 1:20,T cell proliferation was the fastest and about 1.84 times higher than that of imDC.Transfection of miR-let-7i mimic alone significantly promoted T cell proliferation(OD value increased from 1.24 to 1.71),while co-transfection of OE-CCR7 with miR-let-7i mimic significantly reverted to the effect of miR-let-7i mimic alone.After transfection of miR-let-7i inhibitor into cells,T cell proliferation was significantly inhibited(OD decreased from 1.31 to 1.02),and low expression of miR-let-7i while knocking down CCR7 expression significantly reversed the effect of transfection of miR-let-7i alone.In contrast,overexpression of CCR7 alone significantly inhibited the proliferation of T lymphocytes(OD decreased from 1.29 to 0.61)，while the addition of Toll-like receptor signaling pathway agonist significantly reverted the effect of overexpression of CCR7 alone.Inhibition of CCR7 expression alone significantly promoted T-cell proliferation(OD decreased from 1.30 to 1.43),while addition of Toll-like receptor signaling pathway inhibitor reversed the effect of low expression of CCR7 alone.5.Flow cytometry was performed to detect T-cell apoptosis,which was significantly inhibited during LPS-induced DC maturation.While transfecting miR-let-7i mimic alone into cells,apoptosis of T lymphocytes was inhibited again;at this time,after elevating the expression of CCR7 similarly,apoptosis of T lymphocytes was promoted.Transfection of miR-let-7i inhibitor into cells significantly reversed the LPS-induced effect and promoted T cell apoptosis;at the same time,lowering the expression of CCR7 inhibited the effect of low miR-let-7i expression.When the expression of CCR7 alone was elevated,promoting apoptosis of T lymphocytes,the addition of Toll signaling pathway agonist reversed the effect of overexpression of CCR7.T-cell apoptosis was inhibited when CCR7 expression was suppressed alone,and the addition of Toll signaling pathway inhibitor partially reversed the effect of low expression of CCR7.6.After LPS induction,IL-2,IL-4,IL-10,and IFN-γ expressions were 164.31±7.73,82.21±2.84,128.80±7.61,and 195.48±5.14 pg/mL,respectively,which were significantly higher compared with the imDC group.While transfection of miR-let-7i mimic into the cells,IL-2,IFN-γ,IL-4,IL-10 expression was 206.54±4.15,244.85±3.37,54.06±5.68,60.82±4.97 pg/mL,respectively,which significantly promoted the effect of LPS.Co-transfection of miR-let-7i mimic with OE-CCR7 for IL-2,IL-4,IFN-y,and IL-10 expression was 72.66 ±4.15,126.1 1±0.23,120.96±0.49,and 75.71±4.72 pg/mL,respectively,significantly replying to the effect of miR-let-7i mimic transfection alone.While transfection of miR-let-7i inhibitor into cells resulted in IL-2,IFN-γ,IL-4,IL-10 expression of 92.49±4.66,150.54±5.92,106.94±4.67,122.61±4.78 pg/mL,respectively,while simultaneous transfection of si-CCR7 into cells significantly reversed the effect of transfection alone miR-let-7i inhibitor.Transfection of OE-CCR7 alone resulted in down-regulation of IL-2 and IFN-γ expression and significant up-regulation of IL-10 and IL-4 expression,while the addition of Toll signaling pathway agonist significantly reversed the effect of transfection of OE-CCR7 alone;transfection of si-CCR7 alone resulted in up-regulation of IL-2 and IFN-γ expression and significant down-regulation of IL-10 and IL-4 expression,while the addition of Toll The effect of transfection of si-CCR7 alone was partially reversed by the addition of Toll pathway inhibitor.7.Flow cytometry assay of CD4+CD25+Treg cell differentiation,induced by adding LPS,showed that mDC inhibited CD4+CD25+Treg differentiation by an average of only 20.3%.By transfecting miR-let-7i mimic alone,which promotes LPS induction,CD4+CD25+Treg was again inhibited by an average of 19.3%,while by co-highly expressing CCR7 with it,the miR-let-7i mimic effect was suppressed.Transfection of miR-let-7i inhibitor,which inhibited the effect of LPS,significantly increased the number of CD4+CD25+Treg by an average of 33%,while co-inhibition of CCR7 with it reversed the effect of transfection of miR-let-7i inhibitor alone.Overexpression of CCR7 alone with CD4+CD25+ Treg was inhibited and its effect was reversed with the addition of Toll pathway agonist;low expression of CCR7 alone promoted CD4+CD25+Treg differentiation but its effect was reversed with the addition of Toll pathway inhibitor.8.Upon StarBase query,a binding sequence existed between miR-let-7i and CCR7.After verifying the dual luciferase reporter gene,miR-let-7i mimic was able to significantly inhibit the luciferase activity of CCR7-WT(CCR7 wild type),but had no significant effect on CCR7-MUT(CCR7 mutant),and the results indicated a regulatory relationship.9.mDC significantly reduced CCR7 expression compared to imDC by Western Blot(0.78±0.16 vs.0.52±0.15).Overexpression of miR-let-7i,downregulated CCR7 expression(0.77±0.04 to 0.50±0.07);inhibition of miR-let-7i,CCR7 was upregulated(0.81±0.01 to 1.15±0.15).after LPS induction,TLR4(0.45±0.05 to 1.01±0.10),MyD88(0.41±0.01 rose to 0.76±0.05),NF-kB(0.75±0.03 rose to 1.09±0.06),STAT1(0.65±0.16 rose to 1.09±0.13);while after overexpression of CCR7,TLR4(1.01±0.10 fell to 0.77±0.11),MyD88(0.76±0.05 fell to 0.65±0.07),NF-kB(1.09±0.06 decreased to 0.67±0.02),STAT1(1.09±0.13 decreased to 0.74±0.02)inhibited LPS induction;the addition of Toll pathway agonists significantly reverted the OE-CCR7 effect.Low expression of CCR7,TLR4(0.77±0.11 up to 0.83±0.05),MyD88(0.65±0.07 up to 0.95±0.11),NF-kB(0.67±0.02 up to 0.93±0.04),STAT1(0.74±0.02 up to 0.87±0.05)further promoted LPS induction effect LPS induction resulted in elevated MHC Ⅱ,CD80,CD86 expression,and OE-CCR7 reversed this effect,and the addition of Toll pathway agonist reverted the effect of OE-CCR7;si-CCR7 promoted the effect of LPS,and the addition of Toll pathway inhibitor reversed the effect of si-CCR7.10.By establishing a rhesus allogeneic skin transplantation model,imDC cells transfected with empty vector derived from donor rhesus monkey peripheral blood induced culture and imDC cells transfected with miR-let-7i gene inhibitor and blank control PBS solution were injected subcutaneously at a specific time after skin transplantation,and after transplantation for a certain period of time,the transplanted skin pieces were removed for examination.As detected by RT-qPCR,the expression of miR-let-7i was significantly lower in the imDC group injected with the imDC transfected with the inhibited miR-let-7i gene compared with the blank control;the expression of inflammatory factors IL-2 and IFN-y was significantly lower,and the expression of IL-10 and IL-4 was significantly higher.Flow cytometry revealed a significant increase in the percentage of CD4+CD25+Treg cells.In the tissue biopsy of transplanted skin pieces,miR-let-7i inhibitor could effectively inhibit IL-2 and IFN-γ expression and promote IL-10 and IL-4 expression,thus affecting postoperative Th1/Th2 shift,promoting graft tolerance and alleviating graft rejection.Conclusion:In this study,we validated for the first time the effect of miR-let-7i on the maturation and function of dendritic cells in rhesus monkey peripheral blood in vitro cell experiments,and verified that inhibition of miR-let-7i inhibited Toll-like receptor signaling pathway by targeting upregulation of CCR7 expression to maintain DC immature state and affect the function of DC cells,and for the first time in non-primate rhesus monkeys in vivo experiments verified that inhibition of immature The first in vivo experiment demonstrated that inhibition of miR-let-7i signaling in immature dendritic cells induced hyporesponsiveness to immune rejection after skin grafting in rhesus monkeys. |
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