Co-operation Between Natural Killer Cells And Kupffer Cells In Poly I:C/D-GalN-induced Liver Injury

The role of liver as a major organ of the innate immune system has been increasingly recognized.The liver lymphocyte population is enriched in macrophages(Kupffer cells),natural killer and natural killer T cells,which constitute the innate immune system and play a critical role in immune defense against tumors and microbial pathogen.More and more evidences also suggest the contribution of these innate immune cells in the pathogenesis of hepatitis.Kupffer cells can produce various inflammatory mediators that may damage hepatocytes upon activation by bacterial products,such as endotoxin(lipopolysaccharide,LPS) NKT cells are also pivotal in Con A- and alpha-galactosylceramide(α-Galcer)-mediated liver damage via cytokineds and direct cytotoxicity against hepatocytes.NK cells are another population of innate immune cells in the liver,comprising 30-40%of total intrahepatic lymphocytes in human,and 10-20%in mice.The contribution of NK cells in the pathogenesis of human hepatitis or animal models has been reported,but the role and mechanisms remain to be further investigated.In this study,to induce liver injury,we injected mice intravenously with poly I:C (1ug/mice) and intraperitoneally with D-GalN(10 mg/mice).Then we examined the liver injury by determining serum transaminase ALT levels and liver pathologic changes.By cell depletion,effects of the specific lymphocyte population and cellular interactions in the process of liver injury were investigated.The FACS analysis provided us with the precise information about lymphocyte phenotype,activation and cytokine production.Hepatic NK cells and Kupffer cells were isolated by positive magnetic cell sorting(MACS) according to the manufacyure's protocol,and were coculture in vitro with poly I:C stimulation.Moreover,specific neutralization with antibody was applied in vivo and in vitro to explore the potential mechanisms involved in cell interaction.We used ELISA and real time PCR to examine cytokine levels.Our major findings are shown as followed:1.Treatment with poly I:C induces severe liver injury in D-GalN-sensitized miceIn this study,we found that poly I:C could induce severe liver injury in D-GalN sensitized mice,which differed from the mild liver injury of our previous study, where poly I:C alone was injected.Co-administration with poly I:C and D-GalN induced significant elevation of serum ALT and histological necrosis in the liver,but injection with poly I:C or D-GalN alone did not cause any liver injury.We also found that the same amount of poly I:C/D-GalN did not induce any injury in other organs,such as the lungs,colon,and small intestine.2.Poly I:C/D-GalN-induced severe liver injury is NK cell-dependentSince we previously found that NK cells could be activated by poly I:C in vivo and played important roles in poly I:C-induced mild liver injury,we examined the role of NK cells in poly I:C/D-GalN co-administration model.The results showed that treatment with poly I:C/D-GalN induced the accumulation and activation of NK cells in the liver,and depletion of NK cells before poly I:C/D-GalN administration could significantly prevent liver injury.Further,the severe liver injury in SCID mice triggered by poly I:C/D-GalN proved that NK cells could mediate poly I:C/D-GalN-induced liver injury without the help of B cells and T cells.3.NK cell activation depends on the presence of Kupffer cells in poly I:C/D-GalN-treated miceMacrophages express abundant TLRs that enable them to sense the presence of pathogens,and are pivotal in several murine models of hepatitis.To examine the role of Kupffer cells in this model,we depleted Kupffer cells with clodronate-liposomes and verified the effective depletion using immunofluorescent staining with macrophage-specific mAb anti-F4/80.Poly I:C/D-GalN-induced liver injury was markedly reduced in Kupffer cell-depleted mice,indicating that Kupffer cells were indispensable in this model.Further,depletion of Kupffer cells significantly inhibited the accumulation of NK cells in the liver,and suppressed production of IFN-γby NK cells.These results suggested that Kupffer cells were involved in the accumulation and activation of NK cells induced by poly I:C/D-GalN.4.Poly I:C/D-GalN-induced liver injury results from the synergic effects of NK cell-derived IFN-γand Kupffer cell-derived TNF-αIn accordance to the time course of poly I:C/D-GalN-induced liver injury, cytokine levels in the serum and liver up to 48 hours after co-administration were measured by ELISA and real-time RT-PCR,respectively.The levels of TNF-αand IFN-γin the serum increased significantly at 18 hours post poly I:C/D-GalN administration.IFN-γand TNF-αmRNA levels in liver tissue preceded the serum peak by approximately 14 hours and 16 hours,respectively.To evaluate the roles of increased TNF-αand IFN-γin liver injury,we injected poly I:C/D-GalN into IFN-γ^-/- or TNF-α-neutralized mice.The results showed that both TNF-αand IFN-γwere critical in this injury.Further,as a supplement to previous conclusion that D-GalN-treated mice were more sensitive to TNF-α-mediated liver injury,we proved that D-GalN-treated mice were also very sensitive to IFN-γ-mediated liver injury. Co-administration of IFN-γand TNF-αcould cause much more severe liver injury than either cytokine alone,suggesting that TNF-αand IFN-γhad a synergic effect on liver injury.The study demonstrated that after poly I:C/D-GalN injection,IFN-γwas mainly produced by liver NK cells,and NK cell depletion could significantly inhibit IFN-γproduction in the serum.The immunofluorescent staining of liver tissues showed that TNF-αwas produced mainly by Kupffer cells.Similarly,Kupffer cell depletion could markedly inhibit TNF-αproduction in the serum.5.Cross talk between NK cells and Kupffer cells occurs via NKG2D-Rae 1 recognition after being triggered by TLR3 activationWe attempted to determine whether NKG2D-ligand interactions were involved in the cooperation between NK cells and Kupffer cells.Expression of Rae 1 and Mult 1,two important ligands of NKG2D,in the liver was investigated by RT-PCR. Rae 1 expression was markedly increased after poly I:C/D-GalN injection,while Mult 1 expression was not changed.We further found that Rae 1 expression was significantly up-regulated on the surface of Kupffer cells but not on hepatocytes. Poly I:C/D-GalN injection did not significantly affect NKG2D expression on NK cells.To investigate whether the interaction between Rae 1 and NKG2D contributed to the liver damage,we injected mice with NKG2D blocking mAb before poly I:C/D-GalN treatment.Blockade of NKG2D recognition alleviated poly I:C/D-GalN-induced liver injury,and reduced levels of IFN-γin the serum.The in vitro data also showed that blockade of NKG2D recognition reduced IFN-γsecretion by NK cells when co-incubated with poly I:C-stimulated Kupffer cells. 6.IL-12 and IL-18 are also involved in the cross talk between NK cells and Kupffer cellsThe critical roles of IL-12 in poly I:C-induced NK cell-mediated liver mild injury have been demonstrated in our previous study.Here,we observed that both IL-12 and IL-18 were expressed constitutively in the liver,but markedly increased 12 to 18 hours after injection.Modest induction of IL-18 and IL-12 in the serum was observed 18 hours and 12 to18 hours after injection,respectively.Moreover, blockade of either of the two cytokines in vitro reduced IFN-γproduction by NK cells if co-incubated with poly I:C-stimulated Kupffer cells.Conclusion:the present work describes an NK cell-mediated murine model of fulminant hepatitis which is a valuable supplement to established Kupffer cell- or NKT cell-mediated hepatitis(e.g.,LPS/D-GalN model and Con A model).We also demonstrate that the reciprocal activation of hepatic NK cells and Kupffer cells initiates the liver injury in poly I:C/D-GalN-induced hepatitis.To our knowledge, this is the first report depicting cellular cross talk between hepatic NK cells and Kupffer cells mediated by NKG2D-Rae1 recognition.Taken together,our findings may provide insight into innate immune recognition,which is pivotal in acute liver injury;further,our results may aid in investigating potential therapeutic strategies against innate immune-mediated injury in disease settings.