The Role Of Liver NK Cell In Virus-induced Liver Failure

【BACKGROUND&OBJECTIVE】In developing countries and the Asia-Pacific region, hepatitis B virus (HBV) infectionis seriously epidemic. In China. the incidence rate of HBV infection is as high as 10%.Fulminant hepatic failure (FHF) and acute-on-chronic liver failure (ACLF) due to HBVinfection are the most common severe diseases requiring immediate hospitalization inChina and many other Asian countries. Due to the lack of effective clinical treatment.unless an emergency liver transplant, the majority of patients are with poor prognosis. Thepathogenesis of virus-induced fulminant hepatic failure is complicated. Our previousstudy demonstrated that the fibrinogenlike protein-2 (fgl2) prothrombinase expressed byactivated macrophages (Kupper cells) played a crucial role in the pathogenesis of routinehepatitis virus strain 3 (MHV-3)-induced FHF and HBV-induced ACLF (HBV-ACLF).However, another important pathological feature in this disease-hepatic inflammatory cellinfiltration should not be neglected. Substantial accumulated inflammatory cells, on theone hand, secrete large amount of inflammatory mediators, causing the deterioration ofthe liver micro-environment, on the other hand, can directly damage hepatocytes, leadingto hepatocyte apoptosis and necrosis. So it is vital to figure out the role of a variety ofimmune cells in the hepatocytes injury and the mechanism of interaction among immunecells, which help to comprehensively understand the pathogenesis of virus-induced liverfailure and provide important molecular targets for the treatment and the drug researchand development.So far, immunological mechanism in hepatitis B virus infection mostly focuses on acquired immune. Large amounts of data showed that the HBV itself does not directlycause damage to the liver. Rather. the host immune response to the virus plays a key rolein the pathogenesis; especially the antigen-specific cytotoxicity T lymphocyte (CTL)encoded by HBV might be a decisive factor in the virus clearance and the diseaseoutcome. However, the role of innate immune system in virus hepatitis remains obscure.As a major component of the innate immune system, the role of NK cells in viralhepatitis has been documented by many scholars. Accumulating evidence showed thatNK cells played a key role in resisting the virus invasion, inhibiting viral replication andimmune-mediated hepatocyes injury. In addition, compared to the peripheral lymphaticsystem, NK cells are abundant in the liver and account for around one-third ofintrahepatic lymphocytes, implying the special role of this cell type in liver biology.Increasing evidence has implicated the role of liver NK cells in liver damage. Liver NKcells kill freshly isolated hepatocytes by a TRAIL/TRAIL receptor (TRAILR) pathway inpolyinosinic-polycytidylic acid (poly I: C) induced mouse liver injury and induce severeliver injury through NKG2D-NKG2D ligand (NKG2DL) recognition after low-doseConA stimulation in HBV transgenic mice. These findings can help to understand the roleof liver NK cells in the pathogenesis of FHF, but the liver injury in these models aremostly induced by chemical agents, or lack of a natural process of virus infection andreplication. So to further investigate the role of liver NK cells in the virus- induced liverfailure, a mouse model of FHF induced by MHV-3 was established and patients withHBV-ACLF were also investigated.Therefore the purposes of this study are as the follows:1. To establish a mouse model of FHF by peritoneal injection with 100 PFU MHV-3.①To analyze the dynamic change of the proportion and number, activity, cytotoxicity.and the major cytokine production of liver NK cells in the progression of MHV-3induced FHF.②To study the cytotoxicity of liver NK cells against MHV-3-infected hepatocytes.③To investigate the possible molecular mechanism involved in the liver NK cell-mediaed hepatocytes injury in MHV-3 induced FHF.④To investigate the role of CXC chemokine receptor 3(CXCR3)-associatedchemokines (CXCL9/Mig. CXCL10/IP-10) in the migration of NK cells in MHV-3-induced fulminant hepatic failure.2. To investigate the role of liver NK cell in HBV- ACLF.①To comprare the difference of NK cell frequency in the livers of patients with CHBand ACLF by immunohistochemistry staining.②To compare the difference of the NCR (NKP30 and NKP46) expression on theperipheral NK cells from patients with CHB and HBV-ACLF by a multi-colorflow-cytometry assay【METHODS】1. A mouse model of FHF was established by peritoneal infection with 100 PFU MHV-3.The pathological change of live tissue was observed by HE staining, and the levels ofserum AST and ALT were also detected.2. The proportion and number of NK cells in liver, spleen, blood and bone marrow andthe expression of CD69 on liver NK cells at 0, 24, 48 and 72 h post MHV-3 infectionwas analyzed by flow cytometry. The cytotoxic activity of liver NK cells against aNK-sensitive cell line YAC-1 was detected by a non-radioactive cytotoxicity assay.The lever of IFN-γ, TNF-α, and perforin produced by hepatic NK cells was alsodetected by intracellular cytokine staining.3. In vitro hepatic NK cell cytotoxicity assay was performed to assess the cytotoxicity ofliver NK cells against MHV-3-infected hepatocytes.4. An antibody-independent inhibition of hepatic NK cell cytotoxicity assay was used tofurther determine the possible mechanism involved in the liver NK cells-mediatedhepatocytes injury.5. The proportions and numbers of NK cells in liver, spleen, and blood as well as theexpression of CXCR3 on NK cells at Oh and 48h post MHV-3 infection was analyzedby flow cytometry. The mRNA lever of the CXCR3-associated chemokines (Mig,IP-10) in liver at 0h, 24, 48 and 72 post infection was detected by realtime PCR. Atranswell migration assay was used to asses the chemotactic effect ofMHV-3-infected hepatocytes on the spleen lymphocytes and the role of IP-10 in themigration of lymphocytes from spleen.6. Immunohistochemistry was used to analyze the frequency of NK cells (CD3~-CD57~+) in livers of patients with HBV-ACLF and CHB.7. A multicolor flow cytometric analysis was used evalue the expression of two naturalcytotoxicity receptors (NKP30 and NKP46) on peripheral NK cells of patients withHBV-ACLF and CHB.【RESULTS】1. Through HE staining, extensive inflammatory cell infiltration and hepatocyte deathwere observed in the hepatic lobulbe in MHV-3-induced FHF in Balb/cJ mice. Thelevel of serum ALT an AST gradually elevated, with the highest level at 72 hours postMHV-3 infection.2. Post MHV-3 infection, the proportion and number of NK cells in the liver markedlyincreased and peaked at 48 h post-infection compared with normal control (43.2% vs10.02%, 7.11×10~4 vs 1.34×10~3) and remained high thereafter. In contrast, theproportion and number of NK cells in spleen (4.32% vs 5.72%, 1.68×10~5 vs 6.01×10~5) and BM (0.74% vs 2.27%, 7.4×10~4 vs 5.68×10~5) dramatically decreased.Though the proportion of peripheral NK cells increased significantly (18.01% vs4.82%). the number decreased markedly (9.94×10~3 vs 2.46×10~4). These resultssuggested that NK cells migrate to liver from the blood, spleen and bone marrow postMHV-3 infection.3. The proportion of liver NK cells expressing CD69 increased significantly (5.9±1.7%at 24 h; 80.3±6.3% at 48 h; 95.5±3.1% at 72h), suggesting a rapid and dramaticactivation of these cells. Higher levels of serum ALT and AST and severe hepatocyteinjuries were observed at 48-72 h post-infection, indicating a close associationbetween liver NK cell activation and liver dysfunction.4. Intracellular staining found that IFN-αand TNF-αexpression was increased in liverNK cells at 48 h post-infection (4.44% vs 0.11%, 20.91% vs 3.72%). Notably, theincrease was greater for TNF-αthan for IFN-γ, suggesting that TNF-αplays a moreimportant role in MHV-3-induced FHF. In addition, litter expression of perforin byliver NK cells was observed post infection, implying the minimal contribution ofperforin-mediated lysis to hepatocyte necrosis and to subsequent FHF induced byMHV-3. 5. 48h Post MHV-3 infeciton, the cytotoxicity of liver NK cells again YAC-1 cells wasdramatically enhanced.6. The expression of the FasL on liver NK cells dramatically increased (27.17% vs7.78%), whereas the expression of NKP46, NKG2D, and Trail was not upregulated48h post-infection. But notably, the expression of two NK cell natural cytotoxicityreceptors (NKP46 and NKG2D) remained at relative high lever post-infection.Immunohistochemistry found an increase in Fas expression on hepatocytespost-infection. These results strongly suggest the possiblity that a Fas-FasL pathwaytriggered by liver NK cells may contribute to hepatocyte necrosis in MHV-3 inducedFHF.7. To further determine the mechanism of toxicity, two neutralizing antibodies(anti-FasL and anti-NKG2D mAb) were examined in a hepatocyte cytotoxicity assay.Liver NK cells 48 h post-infection were used as effectors, and uninfected or infectedhepatocytes 48 h post-infection were used as targets. Treatment with anti-FasL mAbinhibited cytotoxicity directed to hepatocytes. Unexpectedly, treatment withanti-NKG2D monoclonal antibody also inhibited cytotoxicity to some extentpost-infection even though expression of NKG2D was not increased. Moreover, asynergy between anti-FasL and anti-NKG2D monoclonal antibody was noted.8. Following MHV-3 infection, the remarkably increased frequency of NK cellsexpressing CXCR3 in liver were observed post infection. Whereas, in spleen andperipheral blood, the frequency were both significantly decreased. In addition, themRNAs levels of CXCL9/Mig and CXCL10/IP-10 in the liver tissues weresignificantly increased. The transwell migration assay showed that MHV-3-infectedhepatocytes had the ability to attract and recruit the spleen NK cells, and IP-10 play amajor role in the lymphocytes mobilization from spleen.9. NK cells (CD3~-CD57~+) increased dramatically in the livers of patients withHBV-ACLF compared to patients with mild CHB. In addition, expression of NK cellsnatural cytotoxicity receptors (NKP30 and NKP46) on the peripheral NK cells wasalso upregulated in patients with HBV-ACLF. 【CONCLUSION】1. In this study, we for the first time investigated the role of liver NK cells inMHV-3-induced FHF. Our results showed that following MHV-3 infection, thenumber of NK cells in livers of Balb/cJ mice increased remarkably, peaked at 48hpost-infection, and remained at a high level until sacrifice. In peripheral blood, spleen,and bone marrow, this number decreased significantly. Expression of CD69, cytotoxicactivity, and intracellular IFN-γand TNF-αproduction by liver NK cells at 48hpost-infection were all significantly up-regulated. Highly activated liver NK cellswere cytotoxic to MHV-3-infected hepatocytes and this effect was markedly inhibitedby anti-FasL plus anti-NKG2D monoclonal antibodies. These findings help to deeplyunderstand the pathogenesis of virus-induced FHF, and also provide importantmolecular targets for the subsequent intervention treatments.2. The interaction of the CXCR3 and its ligands (CXCL9/Mig, CXCL10/IP-10) plays animportant role in the migration of lymphocyte and subsequent fulminant liver failure,and IP-10 may be crucial for the lymphocytes mobilization from spleen.3. In this study, we for the first time found the accumulation of NK cells in the livers ofpatients with HBV-ACLF, which will provide a new insight into the pathogenesis ofHBV-ACLF.