Involvement Of Kupffer Cells In A Novel Murine Model Of Hepatitis Induced By L18-MDP And D-galactosamine

Background:Fulminant liver injury,also known as acute liver failure(ALF),is a severe emergent clinical syndrome that arises from many causes,such as viruses,drugs and poisons.This disease shows fast onset,rapid progression and low survival rate,manifested as the large number of hepatocellular necrosis and the significant decrease of liver function in a short term.As the disease progresses,ALF may even involve multiple organs and systems and result in portal hypertension and bleeding,cerebral oedema,hepatic encephalopathy,coagulation dysfunction,renal dysfunction,and respiratory dysfunction.However,it is difficult to predict the onset and development of ALF.Furthermore,the unfortunate reality is that pharmacologic approaches or liver transplantation are ultimately insufficient to save the lives of some ALF patients.Therefore,further studies on the pathogenesis of ALF are urgently needed to improve clinical diagnosis and treatment.The liver is an important innate immune organ and it contains an abundance of immune cells.The innate immune cells accumulate in the liver and play a critical role in pathogen resistance.However,the overactivation of immune cells will induce an inflammatory storm and an unexpected attack against hepatocytes,which will result in autoimmune liver damage.The key to realizing the innate immune responses lie in the mutual recognition and interaction of immune-related proteins with conserved microbe-associated molecular patterns(MAMPs).Among them,NOD2 receptors,which is an important member of the NOD-like receptors(NLRs)family,would recognize and bind the bacterial cell wall product muramyl dipeptide(MDP).This ultimately contributes to the activation of immune cells.Some studies have shown that NOD2 receptors are expressed on various immune cells and hepatic parenchymal cells,and that these receptors are closely related to numerous inflammatory diseases in organisms.However,the role of NOD2 receptors in liver diseases is relatively under studied.Further investigations are required to uncover its specific mechanism of action.While exploring the mechanism of NOD2 receptors in liver injury,it is particularly important to establish a mouse model with acute liver injury.In this study,we drew upon methodologies used in past studies and treated the mice with D-galactosamine(D-Gal N)to sensitize the liver.We then induced fulminant liver injury by combining L18-MDP with D-Gal N and explored the role of innate immune cells in the model.Methods:1.The weight-matched mice were randomly divided into(1)a control group,(2)a group that received an intravenous injection of L18-MDP(10ug/mouse),(3)a group that received an intraperitoneal injection of D-GALN(20mg/mouse),and(4)a group that received a co-injection of L18-MDP(10ug/mouse)/D-GALN(20mg/mouse).All mice were killed 16 hours after receiving treatment.The severity of the liver injury was evaluated by serum ALT level and liver tissue H&E staining.The conditions of lungs,small intestines,colons and kidney were also evaluated by H&E staining.2.Flow cytometry was used to detect the proportion,activation and secretion of cytokine IFN-γ of NK,NKT,CD4+T and CD8+T cells of liver tissue among the control group and combined treatment group.3.Mice were pretreated with clodronate-liposomes to deplete Kupffer cells in vivo.After 48 h of pretreatment with PBS or clodronate-liposomes,all mice were administered with L18-MDP/D-Gal N to induce ALF.The effect of Kupffer cells on L18-MDP/D-Gal N induced liver injury was investigated by measuring serum ALT level and liver tissue H&E staining.4.The proportion and number of neutrophils in the liver after L18-MDP/D-Gal N treatment were detected by flow cytometry to investigate if the combination will affect neutrophils aggregation in the liver.In addition,the proportion and number of neutrophils in the liver were detected by flow cytometry after the depletion of Kupffer cells to investigate the effect of Kupffer cells on the aggregation of neutrophils in the liver.5.The changes of tumor necrosis factor(TNF)-α in liver and serum of mice after combined injection were detected by RT-PCR and ELISA.MP6-XT3 antibody was used to neutralize TNF-α in vivo.Meanwhile,the control group was injected with an equal volume of PBS.After 30 min,ALF was induced in both groups by administrating L18-MDP/D-Gal N.The effect of TNF-α on liver injury was evaluated by serum ALT level assayand liver tissue H&E staining.The changes in the levels of TNF-α in serum were detected by ELISA after the depletion of Kupffer cells,and the molecular mechanism of the action of Kupffer cells was investigated.Results:1.A significant elevation of serum ALT and massive liver necrosis was induced after the co-administration with L18-MDP/ D-GALN.However,this was not observed when L18-MDP or D-Gal N was injected alone.Moreover,L18-MDP/D-GALN co-administration did not induce injury in the lungs,kidneys,small intestine and colon of mice.2.There were no significant differences observed between the L18-MDP/D-Gal N treated mice and control mice in terms of the proportion,number and expression of the activation markers(CD69 and IFN-γ)of NK,NKT,CD4+T and CD8+T cells.3.Kupffer cells were depleted with clodronate-liposomes before L18-MDP/D-Gal N treatment.The treatment of L18-MDP/ D-GALN could not induce a significant increase of serum ALT and liver tissue necrosis in KCs-depleted mice.4.The proportion and the number of neutrophils in the liver increased significantly in mice injected with L18-MDP/D-GALN.However,there were no obvious accumulations of neutrophils in the livers of KCs-depleted mice.5.The levels of TNF-α in serum and TNF-α m RNA in liver tissues increased significantly after the administration of L18-MDP/D-Gal N.However,the depletion of KCs could markedly inhibit TNF-α production in the serum.In addition,TNF-α neutralization was able to provide complete protection against L18-MDP/D-Gal N-induced hepatotoxicity.Conclusion:Muramyl dipeptide with a C18 fatty acid chain(L18-MDP)is a NOD2 agonist that can induce a fulminant liver injury which is mediated by Kupffer cells in D-galactosamine(D-Gal N)-sensitized mice.Kupffer cells can promote the accumulation of neutrophils in the liver and can also produce TNF-α to participate in liver injury.