SCIMP Interference In Kupffer Cells Induces Immune Tolerance Of Rats Receiving Liver Allografts

Objective:SCIMP is a membrane protein that interacts with SLP adaptors and C-terminal Src kinase.It is selectively expressed in B cells and APC and plays a role in immune response.This article is aim to investigate whether SCIMP interference of KCs induces the formation of immune tolerance microenvironment in rats with orthotopic liver transplantation and the related mechanisms.Methods:1.We established acute rejection models of orthotopic liver transplantation in rats.The expression and location of SCIMP was detected by WB,immunohistochemistry and immunofluorescence after liver transplantation.The anti-SCIMP mAb(0.35 mg / body)was injected into the portal vein of model rats,and the equivalent Control mAb was injected into the control group to determine serum liver function and pathological changes of liver tissues.Next,the model rats were randomly divided into:SCIMP-siRNA supergroup: SHAM group,the rats were exposed to theportal vein,and treated with mannose-conjugated polymer(SCIMP-siRNA)through the portal vein(2mg/kg,3 days/times,for 2 weeks);LT group,the rats were undergone orthotopic liver transplantation,and treated with mannose-conjugated polymer(SCIMP-siRNA)through the portal vein(2mg/kg,3 days/times,for 2 weeks);Control supergroup: SHAM group,the rats were exposed to the portal vein,and treated with mannose-conjugated polymer(Control-siRNA)through the portal vein(2mg/kg,3 days/times,for 2 weeks);LT group,the rats were undergone orthotopic liver transplantation,and treated with mannose-conjugated polymer(Control-siRNA)through the portal vein(2mg/kg,3 days/times,for 2 weeks).The expression of SCIMP after liver transplantation in each group postoperatively were detected by WB;The pathological changes of liver tissue in rats were detected by HE and the acute rejection activity was judged by using banff schema;KCs were extracted and the number of CD80(+)MHCII(+)KCs in rats was detected by FCM.2.KCs were isolated from wild-type rats after transplantation,and the relationship between SCIMP and MHCII were detected by immunofluorescence and immunoprecipitation.Then,KCs were isolated from the above mentioned groups and co-culture with the naive T cells,and the effect of KCs on the proliferation and differentiation of T cells were detected by CFSE and EdU.Next,KCs were extracted from wild-type rats and were divided into two groups: Scramble group,KCs were transfectedwith scramble lentivirus as control group;SCIMP-shRNA group,KCs were transfected with SCIMP-shRNA lentivirus.LPS(100ng/mL)was used to treat the above groups at 1h,6h,12 h,24h,36 h,48h.The expression of TLR4,MyD88,p-TAK1,p-MMK,p-p38,p-JNK,p-p65,JAK3,JAK1,p-STAT6,PPARγ,Bid,Bax,Cleared-Caspase3,Bcl2 protein levels were detected by WB;the levels of inflammatory factors IL-6,IL-10,IL-12 and H2O2 were detected by ELISA;the Arg1,Retnla,Tim-4,Mfge8,Mertk,CD36,Gas6,Scarf mRNAs were detected by RT-PCR;FCM was used to detect the changes in the number of CD204(+)CD206(+)KCs in rats;TUNEL was used to detect the apoptosis of KCs;the phagocytic ability of KCs in each group on apoptotic T cells were detected;After the use of JAK3 inhibitor(PF-06651600)/STAT6 inhibitor(As1517499),the expression of JAK3,p-STAT6,PPAR γ,CD206,Tim-4,Mfge8,Gas6 protein levels were detected by WB.3.In vivo,the rat models were established according to the groups in the part of method 1.TUNEL was used to detect the apoptosis of liver tissue after operation;the changes of ALT and AST levels in peripheral serum of rats were detected;the expression level of HGF in liver tissue was detected by immunohistochemistry;the survival time in other rats were observed.Results:1.In vivo,SCIMP expression gradually increased with time,andreached a peak 9 days after surgery,and SCIMP was selectively expressed in KCs.After blocking the function of SCIMP in KCs,the serum liver function of SCIMP mAb group gradually returned to normal levels,and the pathological changes of the liver tissue were close to the normal liver.However,the Control mAb group showed acute rejection.After interfering with SCIMP,the expressions of pro-inflammatory factors IL-6 and IL-12 in the SCIMP-siRNA group were close to normal levels,the levels of the anti-inflammatory factors IL-10 were significantly increased,and the pathological changes of liver tissue were close to normal.Moreover,SCIMP-siRNA group showed non-deterministic rejection reactions.The number of MHC II(+)CD80(+)double positive KCs decreased significantly after interfering with KCs SCIMP.2.In vitro,immunofluorescence showed that both MHC II and SCIMP were expressed on the KCs cell membrane.Co-immunoprecipitation further confirmed that SCIMP interacts with MHC II;interference with KCs SCIMP can significantly inhibit the proliferation of T cells;SCIMP interference inhibits the expression of TLR4 / NFκB / MAPK proteins,and the proinflammatory factors IL-6 and IL-12 in KCs were decreased,but anti-inflammatory factor IL-10 was increased;the expression of JAK3,p-STAT6,PPARγ were increased after SCIMP interference in KCs,while JAK1 expression showed no change and the number of activated KCs to M2 type polarization gradually increased with time.KCs with SCIMPinterference enhanced phagocytosis and clearance of apoptotic T cells.The expression of apoptosis-related bridging molecules,such as Tim-4,Mfge8,and Gas6,were elevated;JAK and STAT6 blockers were used to verified that the inhibition of KCs SCIMP enhanced the ability of KCs to eliminate apoptotic cells through the JAK3 / STAT6 / PPARγ pathway.3.In vivo,it was found that the apoptosis of hepatocytes were increased significantly after liver transplantation.However,there was no significant difference between the apoptotic cells in the SCIMP-siRNA group and the control group,and the liver function in the SCIMP-siRNA group returned to a normal level.At the same time,the level of hepatocyte growth factor(HGF)in the SCIMP-siRNA group was increased,and the survival time of the SCIMP-siRNA group was significantly prolonged,and basically exceeded 100 days.Conclusion:SCIMP expression in KCs are increased after liver transplantation,and SCIMP is positively correlated with MHC II;SCIMP interference promotes the polarization of KCs to M2 type by inhibiting TLR4/NFκB/MAPK and activating JAK3/STAT6/PPARγ,reduces the release of inflammatory factors,promotes the ability of KCs to clear apoptotic T cells,improves the pathological changes of liver,and prolongs the rat survival time,which are conducive to the formation of immune tolerance microenvironment after liver transplantation.