Inhibition Of MicroRNA-155Expression In Kupffer Cells Induces Immune Tolerance In Mouse Liver Transplantation

BackgroundToday, liver transplantation has become a lifesaving procedure forpatients with chronic end-stage liver disease and acute liver failure whenthere are no available medical and surgical treatment options. However,acute rejection is still a major cause of early graft loss and a risk factor forlong-term recipient survival post-transplantation. Although many kinds ofimmunosuppressant are used to control rejection, there still are a highpercentage of cases. So inducement of donor-specific transplant tolerancehas been suggested as one of the best potential solutions to prevent immunerejection in solid organ transplantation. Over a decade of research, ourgroup found that Kupffer cells (KCs), the most important residentmacrophages of the liver, played a crucial role in the pathological changesafter liver transplantation and its mechanism may be closely associatedwith modulating Th1/Th2balance. Recently, a few studies reported thatmicroRNA-155(miR-155) plays a pivotal role in regulation of immunefunction. But so far there is no related research in solid organtransplantation, especially in liver. So exploration of the effect of miR-155of KCs in transplant immunity has great significance. PartⅠThe effect of miR-155on antigen presenting function andSOCS1/JAK/STAT inflammatory signals pathway in activatedKupffer cellsObjective: To observe the effect of enhancement and inhibition ofmiR-155expression on antigen presenting function, SOCS1/JAK/STATinflammatory signals pathway in activated KCs, and its effect on T cellproliferation and apoptosis. Methods: Mouse KCs were isolated bycollagenase digestion and density gradient centrifugation methods. Theidentity, function and viability of KCs were identified using CD68immunostaining, swallowing ink and trypan blue dye exclusion test,respectively. The survival time and morphological changes of KCs werealso observed. Then, KCs were transfected with miR-155inhibitor, controlinhibitor, miR-155mimic and control mimic at a concentration of100nmol/L, respectively. The transfection efficiency was identified byfluorescence microscopy. The levels of miR-155and SOCS1mRNA weremeasured by Real-Time PCR and the levels of FasL, SOCS1, JAK2,p-JAK2, STAT3and p-STAT3protein were analyzed by Western Blot. Theexpressions of MHC-II, CD86and CD40on the surface of KCs wereevaluated by flow cytometry (FCM). T cells derived from mice spleen cellswere co-cultured with the4groups of KCs above, T cell proliferation wasmeasured by MTT assay and apoptosis was determined by Annexin V/PIFCM analysis. The density of IFN-γ, TNF-α and IL-10in the supernatant of co-culture was assessed by ELISA. Results: KCs were seen to swallow alot of ink particles. Trypan blue staining showed the vitality of cells in eachgroup were about more than90%. Immunohistochemistry showed that cellsisolated were KCs. Twenty four hours after being transfected with miR-155inhibitor and mimic, more than90%of KCs can be seen obviousluminescence by fluorescent microscope. Real-time PCR results showedthat the relative expression of miR-155in miR-155mimic group is210.67times than those of control group, and the relative expression of miR-155inmiR-155inhibitor group is0.0029times than those of control group. Theexpression of antigen presenting associated molecules on the surface ofKCs such as MHC-II, CD86and CD40in miR-155inhibitor group wassignificantly lower than in control inhibitor group, but these surfacemolecules in miR-155mimic group was significantly higher than in controlmimic group(P<0.05). Western Blot results displayed the inhibition ofmiR-155levels in KCs can up-regulate SOCS1expression, and thensuppress its downstream factor JAK2/STAT3expression andphosphorylation. Nevertheless, enhancement of miR-155levels in KCsshowed opposite results (P<0.05). The purity of T lymphocyte isolatedfrom spleen cells was75.42%, and the composition of CD4positive Tlymphocyte was52.63%. Three days after co-cultured with KCs transfectedmiR-155inhibitor, the levels of T lymphocyte proliferation andpro-inflammatory cytokines secretion were significantly suppressed, but the levels of T lymphocyte apoptosis and anti-inflammatory cytokinessecretion were significantly enhanced (P<0.05). Conclusion: miR-155inhibitor and mimic effectively suppressed and enhanced the miR-155expression in KCs; inhibition of miR-155activity in KCs decreased theirsurface molecules of MHC II, CD86and CD40expression, weakened theirantigen-presenting function, and impacted the SOCS1/JAK/STATinflammatory pathways, leading to the reduction of pro-inflammatorycytokines secretion. Additionally, KCs transfected with miR-155inhibitorco-cultured with T lymphocyte decreased T lymphocyte proliferation andincreased its apoptosis, which may be associated with up-regulation ofFasL expression in KCs. In contrast, enhancement of miR-155expressionin KCs showed opposite results compared with miR-155inhibition.PartⅡThe establishment of mice orthotopic liver transplantationand RNA interference in Kupffer cells modelsObjective: To establish a model of acute rejection of orthotopic livertransplantation (OLT) in BALB/c to C3H inbred mice, and to explore theefficiency of RNA interference via injection of miR-155short hairpin RNA(miR-155-shRNA) lentiviral into recipients through portal vein. Methods:OLT donated by male inbred BALB/c mice was performed on male inbredC3H mice by Kamada's two-cuff technique without liver arterialanastomosis to establish acute rejection model. Recipients were randomly divided into three groups:①normal saline group (NS): injection of0.2mLNS into recipients through portal vein;②control group (Control): injectionof0.2mL negative control lentiviral at a concentration of1×109Tu/mLthrough portal vein;③transfection group (miR-155-shRNA): injection of0.2mL miR-155-shRNA lentiviral at a concentration of1×109Tu/mLthrough portal vein. Then, the operation time of each step and the levels ofmiR-155expression both in recipient liver and KCs were determined byReal Time-PCR at day1,4,7post-tranplantation, respectively. Results: Wehave successfully established a stable model of OLT in BALB/c to C3Hinbred mice by Kamada's two-cuff technique. There was no significantdifference among the three groups of each step operative time (P>0.05).Real-time PCR results showed that the levels of miR-155expression intransfection group decreased time-dependently and were significantlylower than those of NS group and control group at day1,4,7post-transplantation (P<0.05). Conclusion: We have successfullyestablished a model of OLT in BALB/c to C3H inbred mice by Kamada'stwo-cuff technique without liver arterial anastomosis and demonstratedinjection of miR-155-shRNA lentiviral into recipients through portal veincan effectively down-regulation of miR-155expression both in recipientliver and KCs. PartⅢ The effect of inhibition of miR-155in Kupffer cells onacute rejection in mice liver transplantationObjective: To research the effect of miR-155silence in KCs of the liver ofrecipients on acute rejection in mice liver transplantation. Methods: Afterdemonstration of efficiency of RNA interference via injection ofmiR-155-shRNA lentiviral through portal vein, postoperative survival ratewas observed, and liver tissue and blood samples were obtained at day7after surgery. Liver morphological changes and apoptosis levels wereanalyzed by hematoxylin-eosin (HE) and TUNEL staining. Alanineaminotransferase (ALT) and aspartase mainotransferase (AST) levels inplasma were measured with an automatic biochemical analyser. The mRNAand protein expressions of SOCS1, IFN-γ and IL-10in liver tissue weredetected by real-time PCR and western blot, respectively. Plasma levels ofIFN-γ and IL-10were assayed by ELISA. Results: The model of OLT inBALB/c to C3H inbred mice can induce obvious acute rejection during7days after surgery. The7days survival rate in NS group, control group andtransfected group were25.00%,16.67%and91.67%, respectively. Themethod of injection of miR-155-shRNA lentivirus through the portal veinsignificantly improved liver histopathological changes and liver function,prolonged the survival time and reduced acute rejection. Real-time PCR,Western Blot and ELISA results showed that the IFN-γ mRNA and proteinexpression levels in transfection group were remarkably lower in the NS group and control group (P <0.05); In contrast, the IL-10mRNA and proteinexpression levels were much higher in the NS group and control group (P<0.05). Its mechanism may be closely to miR-155inhibition in transfectiongroup increased SOCS1expression, a negative regulation of inflammatoryfactor. These results were consistent with in vitro data. Conclusion: Themodel of OLT in BALB/c to C3H inbred mice was acute rejection model.The inhibition of miR-155expresson in liver tissues and KCs cansignificantly improve liver morphological and its function, and induceimmune tolerance by up-regulating typical Th2-type cytokine (IL-10) anddown-regulation of Th1-type typical cytokine (IFN-γ) gene and proteinexpression, promoting Th1/Th2immune deviation formation.