| BackgroundNowadays, liver transplantation is the most effective treatment option of end-stage liver disease. But acute rejection post-transplant induced graft function loss still didn’t get an ideal prevention protocol. Limited by the shortage of available donor liver globally, induced grafts specific immune tolerance after transplant remains its major importance in the solid transplantation field. Our researches demonstrated that KCs possessed a bipolar character during the process of post-transplant immune response, however, the precise mechanism remain vague. The recently reported enzyme inositol 1(ERE1)-X-box binding protein 1(XBP1) pathway may not only participated in the unfold protein response, but also deeply involved in the regulation of antigen presenting cells (APCs) function, but liver transplantation was not involved. Therefore, explore the exact mechanism of IRE1-XBP1 pathway in KCs in the induced immune tolerance after transplant, may provide novel target and experimental evidences for clinical practice.Part I The effect of IRE1-XBPl pathway on antigen presenting function, polarization mechanism and inflammatory signals pathway in activated Kupffer cellsObjective To explore the effect of IRE1-XBP1 pathway on antigen presenting function, polarization mechanism and inflammatory signals pathway in activated Kupffer cells, and the role of IRE1-XBP1 pathway T lymphocyte proliferation and apoptosis. Methods ① rat KCs were isolated by IV type collagenase digestion and gradient centrifugation methods. ② The identity, function and viability of KCs were identified using CD68/cd163 immunostaining/CONFOCAL, swallowing ink /Latex beads and trypan blue dye exclusion test, respectively.③ KCs were transfected with XBP1-shRNA, Scrambled-shRNA (Ctrl-shRNA), AdV-XBP1 and AdV-Scrambled (AdV-Scrambled) at a concentration of 100nmol/L, respectively.④ The transfection efficiency was identified by fluorescence microscopy and CONFOCAL.⑤ The expressions of MHC-IL CD86, CD40, CD204 and CD206 on the surface of KCs were evaluated by flow cytometry (FCM) and CONFOCAL. ⑥ The mRNA transcription and protein expression levels of XBP1、IL-6、IFN-γ、TNF-α、IL-17、JAK1、 JAK2、STAT1 and STAT3 were evaluated by Real-Time PCR and Western Blot respectively.⑦T cells derived from rat spleen cells were co-cultured within the 4 groups of KCs mentioned above, T cell proliferation was measured by MTT assay/Brdu labeling assay and apoptosis was determined by Annexin V/PIFCM analysis. ⑧ The density of IL-6、IFN-γ、TNF-α、 IL-17 and IL-10 in the supernatant of co-culture was assessed by ELISA. Results① KCs were observed to swallow a lot of Latex beads or ink particles. Trypan blue staining showed the vitality of cells in each group were about more than 90%. ② Immunohistochemistry and CONFOCOL showed that cells isolated were actually KCs.③ 24 hours after being transfected with XBP1 plasmid followed Lipofectamine 2000 protocol, more than 90% of KCs were observed obvious luminescence by CONFOCOL/fluorescent microscope.④ The mRNA and protein level of XBP1 were measured by RT-PCR and WB, those in XBP1-shRNA group were significantly reduced compared with those in the other three groups, while in AdV-XBP1 groups, results demonstrated entirely the opposite tendency (P<0.05). ⑤ The expression of marker molecules on the surface of KCs such as MHC-II, CD86 and CD40 in XBPl-shRNA group were significantly lower (P<0.05), but CD204 and CD206 expression were much higher, compared with the other three (P<0.05). However the expression tendency of these surface markers were shown the opposite results in AdV-XBP1 group (P<0.05). ⑥ WB revealed the XBP1-shRNA could statistically suppress the protein levels and phosphorylation of JAK1, JAK2, STAT1 and STAT3, which involved in the pro-inflammatory cytokines regulation and KCs polarization (P<0.05). But in AdV-XBP1 group, these protein and its phosphorylation were markedly promoted (P<0.05). ⑧ ELISA results collaborated with WB. ⑨ The purity of T lymphocyte isolated from spleen cells was 78.73±4.35%, and the composition of CD4 positive T lymphocyte was 58.84±10.47%.⑩ Three days after co-cultured with KCs transfected with XBP1-shRNA, the levels of T lymphocyte proliferation and pro-inflammatory cytokines secretion were significantly reduced, but the levels of T lymphocyte apoptosis and anti-inflammatory cytokines secretion were remarkably enhanced (P<0.05). Conclusion ① Blockage of IRE1-XBP1 activation could alter the phenotype of active KCs to M2 like-type and attenuated the capacity of antigen present of KCs, while up regulated the expression of IRE1-XBP1 pathway could change the phenotype of KCs to M1 type plus the promotion of antigen present capacity. ② IRE1-XBP1 pathway blockage could suppress the expression and phosphorylation of JAK1, JAK2, STAT1 and STAT3, which alter the secretion property from M1 to M2; but upregulation of IRE1-XBP1 pathway could elevate expression and phosphorylation of JAK1, JAK2, STAT1 and STAT3, promoted the secretion of pro-inflammatory cytokines ③ KCs transfected with XBP1-shRNA, co-cultured with T lymphocyte limited T lymphocyte proliferation and increased the apoptosis rate. But enhancement of XBP1 expression showed the opposite results compared with XBP1-shRNA group.Part Ⅱ The establishment of rat orthotopic liver transplantation and evaluate the targeting of XBP1-shRNA-PFCN vector towards Kupffer cellsObjective To establish a LEWIS-BN rats acute rejection model of orthotopic liver transplantation (OLT). And evaluate the targeting of XBP1-shRNA-PFCN vector towards Kupffer cells. Methods liver donated by LEWIS rats were transplant into BN recipients by modified Kamada "two-cuff" approach to establish acute rejection model. Lipid-wrapped PFCN was obtained by two-step emulsification and binding to XBPl-shRNA through the postitive/negative potential. Recipients were randomly divided into three groups:(1) XBP1-shRNA-PFCN group (PFCN): injection of XBP1-shRNA-PFCN in a ratio of 1 ml/1 OOOg through portal vein, then injected 2mL saline; (2) XBP-1-shRNA-lentiviral group (lentivir): injection of 2mL XBP1-shRNA-lentiviral at a concentration of 1 × 109 Tu/ mL through portal vein; (3) saline group(NS):injection of 2mL NS through portal vein. The mRNA and protein expression in KCs, hepatocyte, spleen and intestinal at 3d and 7d post-transplant were detected by RT-PCR and Western Blot respectively. Results We established a stable model of OLT in LEWIS to BN rat by Kamada’s two-cuff approach. There shown no significant difference among the three groups of each step operative time (P>0.05). The mean diameter of PFCN were 283.78±23.16.3 day after transplanted, XBP1 expression in KCs were both suppressed in PFCN group and lentivir group, and shown similar suppression efficacy(P>0.05). While in hepatocyte and intestinal, XBP1 expression were both suppressed in PFCN group and lentivir group, but lentivir shown more suppression efficacy respectively(P<0.05); at 7d, PFCN demonstrated more suppression of XBP1 in KCs but less in hepatocyte, compared with lentivir group(P<0.05). Lentivir could continuously suppress XBP1 in spleen and intestinal, compared with the other two groups (P<0.05). Compared with NS group, PFCN shown no statistic difference (P>0.05). Conclusion LEWIS-BN rats OLT were successfully established. PFCN demonstrated more KCs targeting and transfection efficacy than lentivirus vector.Part Ⅲ The role of inhibition of IRE1-XBP1 in Kupffer cells on an acute rejection model of liver transplantation in ratsObjective To explore the effect of IRE1-XBP1 in KCs in allograft liver on acute rejection after rats liver transplantation. Methods ① The establishment of LEWIS-BN rat liver transplantation model were described in part II; ② recipients were randomly divided into three groups as followed:XBP1-shRNA group, Scrambled-shRNA (Ctrl-shRNA) group and NS group.③ Blood from Inferior vena cava and tissues from left lobe were obtained at 7d post-surgery. The treatment was referring to Part II.④ Post-transplant survival time were documented; liver function, pathological changes and T cell apoptosis were observed respectively; ⑤ RT-PCR were used to detected the relative mRNA expression of T-bet, RORyt, IFN-y, IL-17 and IL-10; ⑥ Western Blot were deployed to measured the protein levels of CD40, CD86, CD204, CD206, IFN-y, IL-17 and IL-10; ⑦ ELISA were performed to detected the concentration of IFN-y, IL-17 and IL-10 in plasma. Results ① Recipients in XBP1-shRNA groups obtain a prolonged survival time and a better liver function at day 7 after transplant, compared with Ctrl-shRNA and NS group (P<0.05); the pathological changes in XBP1-shRNA group were mild, the mean RAI score was 4.72±0.21, the mean RAI score in Ctrl-shRNA and NS group were 7.94±0.55 and 8.36±0.32 respectively(P<0.05), both demonstrated severe acute rejection; ② The major transcription factor of Thl/Th17 T-bet and RORyt relative mRNA expression in liver allograft were significantly reduced in XBP1-shRNA group(P<0.05), along with IL-17 and IFN-y(P<0.05), compared with the other two groups, however, the IL-10 levels were remarkably increased in XBP1-shRNA group(P<0.05); ③ In XBP1-shRNA group, the protein expression of CD40, CD86, IFN-y, and IL-17 were statistically decreased (P<0.05); while CD204, CD206 and IL-10 protein levels were much higher(P<0.05), compared with Ctrl-shRNA and NS group respectively; ③ The concentration of IFN-y and IL-17 levels in XBP1-shRNA groups were gradually suppressed while those in Ctrl-shRNA and NS group were increased, both in a time-depended manner (P<0.05), but IL-10 in XBP1-shRNA group were higher than the other two groups at 3d and 7d post-transplant (P<0.05). Conclusion The blockage of IRE1-XBP1 could induced immune suppressed microenvironment and amelirated AcR by altered the polarization of KCs from M1-like towards M2-like, and consequently skewed the Th1/Th17 towards Th2/Treg. |
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