The Role And Mechanism Of The Ly6C^high To Ly6C^low Macrophage Transition During The Liver Injury And Repair

The liver injury and repair is an inflammatory process,which results from various etiological causes and is mediated by many kinds of cells in the liver.The cellular character of liver injury and repair is the degeneration,apoptosis and necrosis of hepatic parenchymal cells,and activation of hepatic non-parenchymal cells.Chronic liver injury leads to collagen deposition,liver fibrosis and even cirrhosis.Acute severe hepatic injury can cause acute hepatic failure.These acute or chronic liver diseases are common disease in clinic and seriously threaten human health.Macrophages are essential innate immune cells,which play vital and dual roles during liver injury and repair.Therefore,to explore the role and function of macrophages during the liver injury and repair may provide new targets for many liver diseases therapy.According to their origin and function,hepatic macrophages can be classified into different subsets,which exert very complicate roles during the liver injury and repair.Once liver is injured,the prenatal yolk sac-derived macrophages,namely Kupffer cells（KCs）in liver,are significantly reduced.Meanwhile,a large amount of Bone marrow-derived macrophages（BMDMs）infiltrate into liver and initiate liver inflammation response.Infiltrated BMDMs（IMs）are also remarkably plastic and heterogeneous cell populations,which can either promote tissue damaging or tissue healing depending on the type of injury and the tissue milieu.IMs can be divided further into two subsets based on their differential expression of ly6C.Ly6C^high(ly6C^hi)IMs exhibit a pro-inflammatory and tissue-damaging phenotype,in contrast,ly6C^low(ly6C^lo)IMs show an anti-inflammatory and tissue-protective phenotype.The ratio of ly6C^hi/ly6C^lo can be considered as an important index for evaluating the function of hepatic macrophages.Moreover,upon phagocytosis of apoptotic hepatocytes,ly6C^hi IMs can switch to ly6C^lo IMs.However,the regulation mechanism on the transition of ly6C^hi IMs to ly6C^lo IMs still remains unclear.Therefore,it is valuable to study the role and mechanism on the transition of ly6C^hi IMs to ly6C^lo IMs during the liver injury and repair,because this study may provide new therapy strategies on targeting different macrophages subsets for liver diseases.Our previous studies have shown that transplantation of M1-polarized macrophages improves murine liver fibrosis through recruitment of more monocyte-derived macrophages and expression of high-level MMPs in the whole liver.However,whether the increased fibrosis resolution in M1 cells delivery group is related to the transition of ly6C^hi IMs to ly6C^lo IMs,and the underlying mechanism of the cells transition still need to be further studied.The Notch signaling pathway is a high conserved signaling during evolution,which plays an important role in various cell fate decisions,such as the development,differentiation and functional regulation of myeloid cells.A variety of molecules of Notch signaling pathway are reported to be expressed on activated macrophages,and activated Notch signaling regulates M1/M2 macrophages activation.Our previous studies have shown that Notch signaling pathway mediates macrophage activation and function through CYLD-NF k B during liver fibrosis.However,it is still unclear whether Notch signaling involves in the transition of ly6C^hi IMs to ly6C^lo IMs during liver injury and repair,and whether Notch signaling can be used as the target for liver disease treatment through the regulation of infiltrated macrophages transition.Regarding on these questions,several experiments were performed as follows:1.Infused different polarized macrophages into CCl₄-induced murine liver fibrosis,and detected the apoptosis of HSCs by immunofluorescence（IF）and TUNEL staining.2.Quantified analysis on the phenotype of infiltrating inflammatory cells into injured livers by FACS.3.Sorted liver macrophages by MACS,and detected the expression level of fibrosis-associated molecules by real-time q PCR.4.Analyzed hepatocytes proliferation and liver function by immunohistochemistry staining（IHC）and biochemistry analysis.5.Using CCR2^-/-mouse to block BMDM infiltration or clodronate liposomes for tissue-resident macrophages clearance,combined with CCl₄-induce liver fibrosis model,to analyze the effect of M1 delivery on the different origins of macrophages in fibrotic liver.6.Established the model of CCl₄-induced liver fibrosis in myeloid-specific disruption of RBP-J mice,and detected the phenotype of infiltrating inflammatory cells in fibrotic livers by FACS.7.Established the murine model of liver regeneration after partial hepatectomy（PHx）,and analyzed the dynamic changes of macrophages subsets by IF and FACS analysis.8.Established the model of liver regeneration after PHx in myeloid-specific disruption of RBP-J mice,and detected the degree of liver regeneration by real-time q PCR,IF,IHC and FACS.Results:1.NK cells were recruited and activated,and the expression of TRAIL in the whole liver was increased,which remarkably promoted the apoptosis of HSCs leading to reduced collagen production after the Infusion of M1 cells into fibrotic liver.2.After the Infusion of M1 cells into fibrotic liver,the number of endogenous macrophages recruitment was increased significantly,the cell phenotype was switched from ly6C^hi IMs to ly6C^lo IMs,which possessed the function of restorative macrophages and promoted the collagen degradation in fibrotic liver.3.Meanwhile,after cell delivery,the recruited macrophages promoted hepatocyte proliferation through secreting HGFs and restored liver function in the fibrotic recipient mice.4.The infused M1 macrophages could maintain their phenotype long time,but the numbers of homing macrophages gradually decreased in recipient mice,suggesting that the M1 cells delivery caused the microenvironment change of fibrotic liver at the early phase after cells transplantation.5.Myeloid-specific RBP-J depletion promoted the transition of ly6C^hi IMs to ly6C^lo IMs in murine fibrotic liver,which maybe the cytological mechanism of myeloid-specific disruption of RBP-J ameliorates murine liver fibrosis.6.The liver regeneration after PHx was accompanied by the dynamic changes of macrophages subsets,namely the transition of ly6C^hi IMs to ly6C^lo IMs was also involved in the liver regeneration.Moreover,myeloid-specific RBP-J deletion accelerated the transition of ly6C^hi IMs to ly6C^lo IMs in liver regeneration after PHx.The underlying mechanism was that ly6C^lo IMs promoted the proliferation of vascular endothelial cell for liver regeneration.In summary,our study showed that the ly6C^lo IMs played very important role in during liver injury and regeneration.Myeloid-specific RBP-J deletion might accelerate the transition of ly6C^hi IMs to ly6C^lo IMs,consequently reduced the liver fibrosis,and improved liver functions and promoted liver regeneration.However,the detailed mechanism of Notch signaling on regulation of the ly6C^hi IMs to ly6C^lo IMs transition during liver injury and repair still need to further study.