The Role Of NK Cells And Its Chemotactic Mechanism In Virus-induced Liver Injury

[BACKGROUND&OBJECTIVE]Fulminant hepatic failure (FHF) is a rare and challenging clinical situation characterized by sudden and severe hepatic injury and dysfunction, various symptoms and complications, and a high mortality (40～80%). The incidence and etiology of FHF varies by geographic region. Viral hepatitis and drug or toxin ingestion are the most common causes, and uncontrolled systemic activation of the immune system appears to be a central cause regardless of etiology.NK cells play an important role in the pathogenesis of fulminant hepatitis. As the first cellular responders after viral infection, NK cells provide a first line of defense against viral infection. CD69belongs to the C-lectin type superfamily and it is a functional triggering molecule on activated NK cells and is one of the earliest cell surface activating markers expressed. In viral infection, correlation between NK cytotoxic activity and CD69expression in Rhesus Macaques was found. It was also well shown that CD69up-regulation correlates with NK cytotoxicity and IFN-y production. In our previous study, we have found that the activation of intrahepatic NK cells correlated with massive liver injury in murine hepatitis virus-3(MHV-3) infected Balb/cJ mice, but the role of CD69+NK cells in virus-induced severe liver injury is not clear. In this study, we further focused on the function of CD69+NK cells contributing to the severe liver injury induced by MHV-3.In previous studies, we also detected that the proportion and number of NK cells in the liver were markedly increased and peaked at48h post-infection compared with normal control, and remained high thereafter. In contrast, the proportion and number of NK cells in the spleen and bone marrow (BM) were dramatically decreased. Although the proportion of periprhral NK cells increased significantly, the absolute number decreased markedly. These results suggest that NK cells migrate to the liver from the blood, spleen and BM post MHV-3infection. Nevertheless, the processes underlying NK cell trafficking and migration to liver remain poorly defined. In our previous study, gene chip analysis was used to screen for cell surface chemokine receptors CCR1, CCR5, CXCR3and CXCR4in liver NK cells following MHV-3infection. Then Real-time quantitative PCR was used to investigate mRNA expression levels of the same chemokine receptors after MHV-3infection, at0,24,48, and72h, respectively. Based on the microarrays and Q-PCR results, we propose CCR5acts as a prominent receptor guiding NK cells into MHV-3-infected liver.CCR5is a member of the chemokine receptor subclass of the G-protein-coupled receptor superfamily and expressed on various cell types including NKT cells, CD4+T cells, macrophages, and NK cells.This superfamily is known to regulate the immune response by interacting with any of three chemokine ligands:MIP-1α, MIP-1β, and RANTES, which are also called CCL-3, CCL-4and CCL-5, respectively. A number of studies suggest a role for CCR5in the progression of liver diseases. Although increased hepatic expression of CCR5ligands (MIP-1α, MIP-1β and RANTES) are observed in FHF patient biopsies, the contribution of CCR5ligands or CCR5to FHF development remains unknown. Here, we suggest that CCR5may play an important role in the recruitment of intrahepatic NK cells and subsequent necroinflammation and hepatic failure in MHV-3infection.Therefore the purposes of this study are as the followings: (1)To investigate the role of CD69+NK cells contributed to the murine hepatitis virus strain3induced murine hepatitis.(2)To investigate the role of CCR5and its corresponding chemokines (CCL3/MIP-la, CCL4/MIP-1βand CCL5/RANTES) in the migration of NK cells in MHV-3induced fulminant hepatic failure.[METHODS]1. A mouse model of FHF was established by peritoneal infection with100PFU MHV-3.2. The CD69expression on NK cells in liver, spleen, bone marrow and peripheral blood were determined by flow cytometry. The correlation between the CD69level in hepatic NK cells and liver injury was studied.3. The functional marker (CD107a), activating and inhibitory receptors (NKG2D and NKG2A) expressing on CD69+NK cells and CD69-NK cells were detected by flow cytometry. Pro-inflammatory cytokines (IL-9, IFN-γ and TNF-α) were also investigated by intracellular staining.4. The expression of CCR5on NK cells at0,12,24,48and72h post MHV-3infection in liver, spleen, blood and bone marrow was analyzed by flow cytometry. Double immunofluorescent staining of hepatic CCR5+NK cells was used to evaluate migration of inflammatory cells after MHV-3infection.5. The expression level of CCR5ligands MIP-1α, MIP-1β, and RANTES was measured in liver following MHV-3infection at0h,12h,24h,48h and72h by ELISA and immunohistological staining. The mRNA level of the MIP-1α, MIP-1β, and RANTES in hepatocytes at0h,24h,48h and72h post infection was detected by realtime PCR.6. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes on the splenic NK cells and the role of CCR5and its corresponding chemokines (MIP-1α, MIP-1β, and RANTES) in the migration of splenic NK cells.7. The survival time of adoptive transfer splenic NK cells vial the tail vein with or without CCR5-blocking antibody of MHV-3-infected mice with NK depletion was evaluated. The pathological changes of live tissue were observed by H&E staining, and the levels of serum AST and ALT were detected at24h after adoptive transfer. The absolute counting of migrant splenic NK cells in liver was also evaluated by flow cytometry.[RESULTS]1. The CD69expression on hepatic NK cells markedly increased at48h (61.37±4.18%V.S.0.92±0.12%, P<0.0001) and peaked at72h post-infection compared to the normal control (86.52±2.97%V.S.0.92±0.12%, P<0.0001). Similarly, the frequencies of CD69+NK cells in spleen and BM also increased significantly at48h (26.63±3.40%VS.1.37±0.42%, P=0.0193for spleen and16.97±3.67%V.S.0.80±0.12%, P=0.0454for BM) and peaked at72h post-infection (66.13±6.49%V.S.1.37±0.42%, P=0.0111for spleen and29.13±4.09%V.S.0.80±0.12%, P=0.0191). The CD69expression on NK cells in peripheral blood showed a similar tendency as in liver, with the peak at72h (6.40±0.44%V.S.1.16±0.09%, P=0.0077), but without significant difference at48h.2. The level of ALT in serum dramatically increased at48h and peaked at72h post-infection compared with normal control (11880.0±1174.0IU/mlV.S.23.0±1.53IU/ml, P<0.0001), so as to the AST level (23907±1199.0IU/ml V.S.78.33±8.25IU/ml, P<0.0001). Both ALT and AST levels are highly correlated with the CD69expression on percentage of hepatic CD69+NK cells (r=0.989and r=0.971respectively).3. In hepatic CD69+NK cells, the percentage of CD107a increased at48h (4.67±0.38%, P<0.001) and peaked at72h (12.8±0.8%, p<0.001) after MHV-3infection; NKG2D increased at48h (2.48±0.67%, P<0.001) and peaked at72h (4.18±0.57%, P<0.001) after MHV-3infection. NKG2A expressed by CD69-NK cells increased at24h (0.85±0.21%, P<0.001) and then decreased afterwards. IFN-γ increased at48h (3.2±0.73%, P<0.001) and peaked at72h (3.45±0.93%, P<0.001), meanwhile, TNF-a and IL-9peaked at48h (10.83±0.70%and4.1±1.23%for TNF-α and IL-9, both P<0.001).4. Post MHV-3infection, the frequencies of CCR5+NK cells in the liver increased markedly and peaked at24h compared with normal control (41%P=0.0013). In contrast, the frequency of CCR5+NK cells in spleen and bone marrow peaked at12h post-infection at40%and36%, respectively, and then decreased. Interestingly, CCR5+peripheral NK cells showed the same tendency as seen in the liver, peaking at24h (31%) and decreasing after. FITC-labeled anti-mouse CD3e, or CD49b, and PE labeled anti-mouse CD195(CCR5) were used to stain paraffin-embedded serial liver sections after MHV-3infection. CD3e-CD49b+CCR5+cells represent CCR5+NK cells. In early stage infection (24h), CCR5+NK cells were the predominant inflammatory cells, followed by other inflammatory cells (such as T cells) migrating to liver. These results suggest that CCR5+NK cells migrate from the blood, spleen, and BM to the liver during the early stages of viral infection after MHV-3infection in Balb/cJ mice.5. MIP-1α, MIP-1β, and RANTES expression was detected in liver by ELISA. All three chemokines showed a steady increase, peaking as94ng/ml (MIP-1α),0.8ng/ml (MIP-1β), and160ng/ml (RANTES). Immunohistological staining was to examine chemokine expression at the same time points. Expression of MIP-1α, MIP-1β, and RANTES became confluent at72h after MHV-3infection. MIP-1α、MIP-1β and RANTES mRNA expression from infectious hepatocytes showed an upward tendency up to72h, reaching269x,2015x, and189x relative to baseline. These results suggest that expression of CCR5chemokines in liver increased after MHV-3infection, during which CCR5+NK cells migrate to the liver from peripheral organs.6. MHV-3infected hepatocytes can attract splenic NK cells, and this effect was blocked by anti-CCR5monoclonal antibody. In addition, recombinant mouse MIP-1α, MIP-1β, and RANTES protein can also attract splenic NK cells. MHV-3infected hepatocytes recruit splenic NK cells, and this effect can be blocked by anti-MIP-1α, anti-MIP-1β, and anti-RANTES mAb, respectively, although only the latter two were statistically significant. The transwell migration assay showed that MHV-3-infected hepatocytes had the ability to attract and recruit the splenic NK cells, and CCR5and its corresponding chemokines play a major role in the NK cells mobilization from spleen.7. After adoptive transfer of the splenic CCR5-blocked NK cells vial the tail vein in MHV-3-infected mice depleted of NK cells, the survival time was prolonged to at least84h, one of which survived to90h. Through HE staining, extensive inflammatory cell infiltration and hepatocyte death were observed in CCR5+NK group, and the levels of serum ALT an AST were greater than the CCR5-NK group, so as the absolute counting of migrant splenic NK cells. These results suggest blocking CCR5on NK cells reduces recruitment of NK cells to liver after MHV-3infection, and indicating CCR5plays an important role in recruiting NK cells to liver in FHF.[CONCLUSION]1. After MHV-3infection, the number of CD69+NK cells in the liver of Balb/cJ mice increased markedly and peaked at72h post-infection. Similar changes were also observed in spleen, bone marrow and peripheral blood. Meanwhile, the CD69expression on hepatic NK cells was highly correlated with the serum level of ALT and AST. Expression of CD107a and NKG2D, as well as the production of TNF-a, IFN-y and IL-9by hepatic CD69+NK cells were all significantly up-regulated during48-72h post-infection. In contrast, the NKG2A expression increased in hepatic CD69-NK cells but not CD69+NK cells. These results indicate that hepatic CD69+NK cells play a pivotal role in the pathogenesis of FHF by enhancing degranulation and cytotoxic ability of NK cells and increasing production of pro-inflammatory cytokines.2. Our results showed that following MHV-3infection, CCR5+NK migration from the blood, spleen, and BM to the liver. Hepatic CCR5+NK cells peaked at24h, while the chemokines MIP-1α, MIP-1β, and RANTES continued to increase during infection. Transwell assays revealed decreased NK cell migration towards infected hepatocytes after blocking CCR5on NK cells with monoclonal antibody. Neutralization of CCR5ligands MIP-1β and RANTES, but not MIP-1α, decreased NK cell migration. Infusion of CCR5-blocked NK cells in MHV-3-infected mice depleted of NK cells prolonged survival from72h to90h. These results indicate that CCR5and its ligands MIP-1β and RANTES play a critical role in the hepatic recruitment of NK cells in MHV-3-induced and immune-mediated liver injury.