The Role Of Phenotypic Transformation Of Kupffer Cells Induced By IRE1? In Ischemia Reperfusion Injury Of Fatty Liver

China is a country with high incidence of liver diseases.Liver transplantation(LT)is one of the effective treatments for several end-stage liver diseases,such as cirrhosis,liver failure and liver cancer.In recent years,due to the shortage of liver donors,marginal livers were used more frequently for liver transplantation,and fatty liver is the main source of marginal donors.Along with the continually increasing of the people's life level,the happening rate of non-alcoholic fatty liver disease(NAFLD)becomes higher,which causes more source of marginal donors.However,one problem of using fatty liver as the marginal donor is that fatty liver is more sensitive to ischemia-reperfusion injury(IRI)and there is a high malfunction rate of graft liver after LT.However,no effective method has been developed to alleviate it.As the major inherent immune cells in liver,kupffer cells(KCs)plays an important role during hepatic IRI and the development of NAFLD.KCs can be divided into two types,pro-inflammatory(Ml-type)and anti-inflammatory(M2-type),according to their functions.The acute phase of liver IRI is mainly associated with M1-type KCs.Recent studies found that the M2-type KCs decreased in NAFLD while the M1-type KCs increased.Endoplasmic reticulum stress(ER-stress)is a cellular stress response caused by the accumulation of unfolded proteins in ER lumen.Continuous and serious ER-stress can lead to steatosis,inflammation and even apoptosis.ER-stress is activated in hepatocytes during IRI,and the ER-stress in KCs was also found to be involved in IRI in recent studies.However,ER-stress of KCs in fatty liver is not clear at all.Based on the above background,we focus on the ER-stress of KCs in fatty liver and whether activated ER-stress induces its proinflammatory-phenotypic(M1-phenotypic)shift to aggravate IRI of fatty liver.In this study,mice were fed with high fat diet to induce steatosis.IRI in mice fed with HFD was more severe than control group as well as increased level of ROS and recruitment of inflammatory cells.We extracted KCs from both CD and HFD feeding mice received IRI.ER-stress of KCs was activated more significantly in HFD feeding mice undergoing IRI than CD feeding mice.And the levels of proinflammatory cytokines,such as IL6,IL1? and TNFa,were all higher in HFD feeding mice receiving IRI,suggesting that the inflammation in these mice was more severe.In addition,the ER-stress of KCs was also reduced in the presence of 4PBA,inhibitor of ER-stress,in both CD and HFD feeding mice with IRI.So,IRI activated ER-stress of KCs which induced its Ml-phenotypic shift to aggravate IRI of fatty liver.ER-stress of KCs was activated more significantly in HFD feeding mice undergoing IRI.So,we suspected that ER-stress of KCs was activated in fatty liver and KCs with activated ER-stress secreted more proinflammatory cytokines to induce its M1-phenotypic shift in fatty liver,resulting in more severe IRI.Then we focused on the ER-stress of KCs in fatty liver without IRI.ER-stress of KCs was activated in HFD feeding mice but not in CD feeding mice,especially the IRE la signal pathway.What's more,as the hepatic steatosis became worse,the ER-stress of KCs also increased.Consistent with the ER-stress of KCs,the levels of Ml-type markers and serum pro-inflammatory factors were in parallel with the progression of liver steatosis.What's more,we extracted bone marrow-derived macrophages,(BMDMs)and treated with tunicamycin to induce ER-stress.In line with our expectations,M1 phenotype markers were increased with stimulation of tunicamycin and could be suppressed by 4-PBA.IRE la,the ER-resident transmembrane protein kinase and endoribonuclease,is the most conserved ER-stress sensor.And IRE1? was the most significantly upregulated ER-stress sensor in KCs of CD feeding mice with IRI and HFD feeding mice with or without IRI.So,we next wanted to investigate whether IRE1?participates in governing M1-M2 macrophage phenotypic transformation.We found that BMDMs that with IREla knockdown or pretreated with 4?8C,an IRE1 inhibitor,exhibited reduced pro-inflammatory transformation and enhanced anti-inflammatory transformation with LPS/IFNy or IL4 stimulation.We also found that IRE1?suppression could regulate STAT1 and STAT6 to promote M2-type polarization of macrophage and inhibit M1-type polarization.So,IRE1? plays an important role in governing Ml-M2 macrophage phenotypic transformation.We next investigated whether knockdown of IRE1? in KCs could attenuate IRI of fatty liver.To this end,we first depleted KCs in mice using clodronate liposome,and then the KCs with IRE 1?-knockdown or pre-treated with 4?8c were transfused into the mice via portal vein,followed by IRI.In line with our expectations,IRE1? knockdown could pro-mote M2-type polarization of macrophage,which showed anti-inflammatory effect and attenuated IRI in fatty liver.In summary,our study discovers a new mechanism of why fatty liver is more susceptible to IRI,and demonstrates the immune regulatory role of IRE1? in KCs of NAFLD which may provide a potential target to reduce the fatty liver associated IRI in LT.