| Background:Antigen presenting cells(APCs),especially dendritic cells(DC),have long been considered to play an important role in allograft rejection.Activation of APCs can promote humoral rejection and T cell-mediated rejection,thereby enhancing the acquired immune response.Dendritic cells,as the most powerful antigen presenting cells,are involved in antigen presentation and identification,initiation of immune responses and induction of graft rejection.DC not only secrete TNF-α to promote maturation and antigen presentation,but also produce cytokines such as IL-1β,IL-12,IL-23 to regulate T cell differentiation and activation.In most tissues,DC exists in the immature DC(imDC)state and does not stimulate naive T cells because it lacks the level of helper signals required to activate T cells.Thus,by modulating DC(which inhibit maturation and antigen presentation),they may promote immune tolerance and benefit allografts.Therefore,DC may be a useful target for the prevention and treatment of graft rejection.MicroRNAs(miRNAs)have recently emerged as major regulators of gene expression associated with most biological functions,such as cell proliferation,differentiation,apoptosis,and tumorigenesis.The most studied miRNA in DC is anti-miR-155.Studies have shown that miRNAs may be involved in allograft rejection,but the exact mechanism,especially how miRNAs affect antigen-presenting cells(e.g.,dendritic cells)during rejection,remains to be elucidated.Therefore,science hypothesized that antisense anti-miR-155 could induce immune hyporesponsiveness or immune tolerance by regulating dendritic cell maturation,JAK/STAT/SOCS1 signaling related dendritic cell maturation.Therefore,we investigated the role of antisense anti-miR-155 by inducing rhesus monkey peripheral blood immature dendritic cells to affect the expression of costimulatory molecules,the balance of helper T molecules(Th)1 and 2,and the expression of JAK/STAT/SOCS signaling pathway proteins.In view of the rejection of allogeneic organ transplantation,how to solve chronic rejection,induce immune tolerance,and improve the long-term survival rate of transplanted organs is the most important problem.The advantages of non-primates as model animals compared with traditional rodents and their important applications in the field of life medicine have become increasingly prominent.Organ transplantation based on non-human primates has the unique advantage of similar immune system and homology with humans.The similarity of immunology and physiology between non-human primates and humans makes the non-human primate model of great value in the study of virus pathogenesis,immunity,vaccines and anti-infection efficacy of drugs.The immune system of rhesus monkeys is very similar to that of humans,and they have high homology.For example,human MHC Ⅱand TCR genes show strong conservation in rhesus monkeys[1].Targeting DC to modulate antigen-specific immunity can lead to long-term allograft survival by reducing the development of Chronic allograft vascular disease(CAV)and chronic allograft rejection in solid organ transplantation.The success of organ transplantation began with the skin.In view of the above problems,we plan to start from the non-human primate rhesus monkey skin transplantation,and take rhesus monkey as the object to conduct skin transplantation research,which is innovative to some extent and provides a certain basis for future solid organ transplantation.Objective:This project intends to(1)construct imDC anti-miR-155 and study its effect on DC maturation and apoptosis;(2)To investigate the effects of imDC anti-miR-155 on T lymphocyte proliferation and T cell subsets in vitro;(3)The effect on skin transplantation in rhesus monkey skin transplantation model and in vitro T cell co-culture model;(4)To clarify the mechanism of SOCS1/JAK/STAT in the immune tolerance of imDC anti-miR-155 in rhesus monkeys and investigate the mechanism of imDC anti-miR-155.This project intends to(1)construct imDC anti-miR-155 and study its effect on DC maturation and apoptosis;(2)To investigate the effects of imDC anti-miR-155 on T lymphocyte proliferation and T cell subsets in vitro;(3)The effect of imDC anti-miR-155 on skin transplantation in rhesus monkey acute rejection model and in vitro T cell co-culture model;(4)To clarify the mechanism of SOCS1/JAK/STAT on imDC anti-miR-155 in rhesus monkey skin graft immune hyporeactivity study.Methods:1.Construction,culture and identification of imDCanti-miR-155Mononuclear cells were isolated using Peripheral blood from rhesus monkeys.The differences of imDC,mDC and imDC anti-miR-155 were observed by GM-CSF、IL-4 and TNF-α culture and induction of imDC.ImDC were infected with antisense miR-155 lentivirus in vitro,and the expression of anti-miR-155 in imDC induced to become mDC and imDC after infection was detected by qPCR.The effect of anti-miR-155 on the morphology of imDC and mDC was observed by inverted microscope,scanning electron microscope and transmission electron microscope.The effects of anti-miR-155 on DC phenotype and DC apoptosis were detected by flow cytometry.The secretion levels of IL-2,IL-4,IL-10 and IFN-γ in the supernatant of cell culture medium were detected by Elisa.2.The functional effect of imDC anti-miR-155 on T cellsT cells were co-cultured with imDC anti-miR-155 in Transwell chamber;they were grouped into T cells,T cells+mDC,and T cells+imDC anti-miR-155.The effect of imDC anti-miR-155 on T cell proliferation was detected by mixed lymphocyte assay;the effect of imDC anti-miR-155 on T cell apoptosis was detected by flow cytometry;the induction effect of imDC anti-miR-155 on Treg was detected by flow cytometry;The secretion levels of IL-2,IL-4,IL-10 and IFN-γ in the culture supernatant of DC.The expression levels of related biomarkers of Th1,Th2,Th17 and Treg were detected by qPCR and western blot.The expression of Treg biomarker Foxp3 and regulatory factor MyD88 was detected by immunofluorescence.3.Effect of imDC anti-miR-155 on acute immune rejection after skin allograft in rhesus monkeysThe ABO blood type was detected,and the patients were divided into control group,imDC anti-NC group and imDC anti-miR-155 group,given treatment factors,and performed allogeneic skin transplantation.The basic vital signs,spirit and appetite were observed,and the skin graft was observed every other day;the graft skin biopsy was performed 7 days after the operation,and the degree of rejection was determined by acute rejection score and HE staining.On the 7th day after the operation,flow cytometry was used to detect the apoptosis of CD4+T cells in peripheral blood of rhesus monkeys after skin transplantation and the changes in the ratio of CD4+CD25+FOXP3+T cells in peripheral blood;Foxp3,c-Fos,GATA,MyD88,RORyt,TAB2,T-bet,TLR4 mRNA expression levels in peripheral blood were detected by qPCR and western blot.The changes of cytokines IL-2,IL-10,IL-4 and INF-γ were detected by Elisa.The expression level and fraction of Foxp3 protein were detected by immunofluorescence.4.Mechanism of imDC anti-miR-155 on TregsThe targeting relationship between miR-155 and SOCS1 was predicted by bioinformatics;.The changes of SOCS1 during DC maturation were detected by qPCR and immunofluorescence.Design and synthesis of SOCS1 targeting oligonucleotide sequences;The expression of SOCS1 in skin grafts was detected by immunohistochemistry.The overexpression plasmid(OE-SOCS1)and siRNA(OE-SOCS1)of SOCS1 were designed and synthesized,and the complement experiment was carried out.The phenotype and apoptosis of imDCanti-miR-155 were detected by flow cytometry.The secretion of IL-2,IL-4,IL-10 and IFN-y in cell culture supernatant was detected by Elisa.QPCR and Western blot were used to detect the expression changes of related markers of Th1,Th2,Th17 and Treg,and related genes and proteins in JAK/STAT signaling pathway.Immunofluorescence was used to detect the expression of Treg markers Foxp3 and SOCS1.Results:1.Construction,culture and identification results of imDC anti-miR-155 in rhesus macaquesThe expression level of anti-miR-155 was significantly increased during DC maturation.imDCanti-miR-155 was successfully constructed,and its mRNA expression level of anti-miR-155 was significantly inhibited.Whether induced or not,imDC anti-miR-155 cultured in vitro was similar to imDC,with uneven surface,shallow folds and fewer burrs,while mDC had many dendritic protrusions on the surface,which were large in size and varied in length and morphology.Flow cytometry showed that imDCanti-miR-155 was similar to imDC,with low expression of CD80,CD83,MHCII and high expression of CD1α,while high expression of CD80,CD83,MHCII and low expression of CD1α in mDC.There was no significant difference in apoptosis rate between imDC,mDC and imDCanti-miR-155.The levels of IL-4 and IL-10 in imDC anti-miR-155 culture supernatant were higher than those in LPS-induced mDC culture supernatant.This suggests that imDC anti-miR-155 shows some tolerance potential.2.Functional effects of imDC anti-miR-155 on T cellsT cells were co-cultured with imDCanti-miR-155 in Transwell chamber.They were divided into T cells,T cells+mDC,and T cells+imDCanti-miR-155.Flow cytometry showed that the apoptosis level of imDCanti-miR-155+T cells was higher than that of mDC+T cells.Mixed lymphocyte assay showed that mDC could promote T cell proliferation compared with control group.imDCanti-miR-155 could inhibit the proliferation of T cells.It was further found that imDCanti-miR-155 could increase the proportion of co-cultured Tregs,up-regulate the contents of IL-10 and IL-4 in co-culture supernatant,and down-regulate the contents of IL-2 and IFN-y.QPCR and Western blot results showed that imDCanti-miR-155 could up-regulate the expression levels of GATA-3,FoxP3,TGF-β,and down-regulate the expression levels of T-bet and RORγt.Meanwhile,imDCanti-miR-155 can inhibit the expression of inflammatory factors in MyD88/TLR4 signaling pathway.3.Effects of imDCanti-miR-155 on immune hyporesponsiveness and T cell subsets in rhesus monkeys after acute rejection of skin allograftsTwo days before skin transplantation,different imDC suspensions(1×106/ml)were intravenously injected.Medium-thickness skin grafts were performed on the day of experiment,and imDC suspensions of different treatment groups(1×106/ml)were injected under the skin grafts and intravenously on the day of surgery.Rhesus monkeys in each group had good vital signs,spirit and appetite.Compared with the imDC group,the survival time of grafts in donor-derived imDCanti-miR-155 was significantly longer than that in imDC anti-NC and donor-derived imDC groups.HE staining showed that the degree of cell rejection in the imDC anti-miR-155 group was significantly less than that in the imDC and imDC anti-NC groups.Skin tissue sections showed clear skin tissue structure,mild infiltration of local inflammatory cells and micro-angiogenesis,indicating good growth and healing of the allograft skin.On the 7th day after operation,the proportion of apoptosis and Tregs in peripheral blood T cells in imDC anti-miR-155 group were significantly higher than those in imDC group and imDC anti-NC group.In contrast to the imDC and imDCanti-NC groups,the levels of proinflammatory factors IL-2 and IFN-y in peripheral blood of imDC anti-miR-155 group decreased after skin transplantation,while the levels of IL-4 and IL-10 increased.On the 7th day after operation,qPCR and Westerm blot results showed that the mRNA and protein expression levels of TLR4,MyD88,RORyt,TAB2 and T-BET in imDC anti-miR-155 group were lower than those in imDC and imDCanti-NC groups.The mRNA and protein contents of TGF-(3,c-fos,FOXP3 and GATA-3 were significantly increased compared with those before transfection.Similarly,the immunofluorescence intensity of FoxP3 in imDCanti-miR-155 group was significantly higher than that in imDC and imDC anti-NC groups,while MyD88 was the opposite.4.The role of SOCS1/JAK/STAT in immune tolerance of imDC anti-miR-155By using Targetscan and other bioinformatics software,we predicted that SOCS1 was the direct target of anti-miR-155.The dual luciferase reporter assay verified the targeting relationship between anti-miR-155 and SOCS1.SOCS1 was previously found to be highly expressed in imDC anti-miR-155 cells in vitro and in animal model grafts.Therefore,we used the remediation experiment to verify the effect of SOCS1 on DC and Treg in anti-miR-155.The results showed that siSOCS1 down-regulated the mRNA and protein expressions of FoxP3,T-bet and TGF-β,up-regulated the mRNA and protein expressions of GATA-3 and RORγt,and inhibited the expression of JAK/STAT signaling pathway molecules.Increasing SOCS1 by imDC anti-miR-155 can reduce the expression of STAT1 and STAT6,reduce the production of IFN-γ,and induce Th2 cell response.Immunofluorescence further verified that siSOCS1 rescued the fluorescence intensity of Foxp3 enhanced by anti-miR-155.Conclusions:1.Anti-miR-155 can inhibit DC maturation,maintain imDC immature state under inflammatory background,stimulate T lymphocyte proliferation,inhibit Thl proinflammatory factors,enhance Th2 anti-inflammatory response,and induce a certain degree of immune hyporesponse.2.The inflammatory molecules related to TLR4,MyD88 and TAB2 in the Toll-like signaling pathway of imDC anti-miR-155 decreased compared with the control group.imDC anti-miR-155 induced Treg cell proliferation,promoted Treg differentiation and decreased Th1 and Th17 cell secretion.Enhance the anti-inflammatory effect of Th2.3.imDC anti-miR-155 prolonged the survival time of rhesus monkey skin grafts,significantly alleviated graft vascular lesions and inflammatory cell infiltration,and alleviated acute immune rejection after skin grafts.imDC anti-miR-155 may alleviate the rejection of skin grafts in rhesus monkeys by attenuating the immune effects of Th1 and Th17,and prolong the survival time of grafts.4.SOCS1 is a regulatory target gene of miR-155.Anti-miR-155 inhibits the activation of JAK/STAT signaling pathway by regulating the expression of SOCS1,thereby promoting T cell apoptosis,inducing Treg cell proliferation and other biological effects,inhibiting Thl cell differentiation and promoting Th2.Finally affect the occurrence and development of acute immune rejection.imDC anti-miR-155-mediated low immune response may be related to SOCS1/JAK/STAT signaling pathway.Therefore,it is speculated that maintaining the immature state of immature dendritic cells and inducing Treg cells by anti-miR-155 may be the possible mechanism to reduce the acute inhibitory rejection and enhance the anti-inflammatory effect. |
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