The Role Of Hepatic Myeloid-derived Suppressor Cells In Virus Induced Acute Liver Failure

[B ACKGROUND&OB JECTIVE]In developing countries and the Asia-Pacific region, hepatitis B virus (HBV) infection is seriously epidemic. In China, the incidence rate of HBV infection is as high as10%. Fulminant hepatic failure (FHF) and acute-on-chronic liver failure (ACLF) due to HBV infection are the most common severe diseases requiring immediate hospitalization in China 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. The liver is enriched in natural killer (NK) cells that play a critical role in first-line immune defense against invading pathogens, modulation of liver injury, and recruitment of circulating lymphocytes. Recent studies have clearly suggested that NK cells contribute to immune-mediated liver injury in mouse models and in patients with hepatitis C virus (HCV) or hepatitis B virus (HBV) infections. In murine cytomegalovirus (MCMV) and murine hepatitis virus-3(MHV-3) infection models, NK cells contraction has been observed. After virus infection, the role of suppressor cell populations towards NK cell activity is largely unknown, especially at the innate immune stage.Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells endowed with suppressive activity. MDSCs expand and acquire suppressive functions under pathological conditions, including cancer, acute and chronic infections, trauma, transplantation, diabetes, and some autoimmune diseases MDSCs are defined in mice on the basis of CDllb and Gr-1marker expression and the functional ability to inhibit T-lymphocyte activation. However, positive CD11b and Gr-1staining is not unique to MDSCs and not all CDllb+Gr-l+cells are immunosuppressive. The population of CD11b+Gr-1+cells is therefore not fully equivalent to MDSCs, as it also includes mature neutrophils, inflammatory monocytes, tumor necrosis factor and inducible nitric oxide synthase-producing dendritic cells (Tip-DCs), and immature myeloid cells. Evidence accumulated so far indicates that MDSCs are not simply activated inflammatory monocytes or neutrophils. MDSCs are also characterized by their potent ability to suppress different aspects of immune responses, especially T-cell proliferation and cytokine production. The mechanisms include upregulation of arginase, cyclooxigenase-2, prostaglandin E2, NO, reactive oxygen species (ROS), regulatory T cells as well as production of TGF-β, depletion of cysteine, and downregulation of T-cell L-selectin expression. These mechanisms are described in a number of recent reviewsThe cross-talk between MDSCs and NK cells is still controversial in regards to cancer. Several studies have demonstrated that MDSCs inhibit NK cell cytotoxicity against tumor cells and block NK cell production of IFN-y. These activities require cell contact between the MDSCs and target cells. However, another study has demonstrated that Gr-1+CDllb+F4/80+MDSCs, which suppressed T-cell activation, express retinoic acid early inducible-1(Rae-1) and can activate NK cells. Activated NK cells, in turn, can eliminate MDSCs.There has been little exploration of cross-talk between these two immune cell populations in regards to virus infections. In this study, evidence is provided that MDSCs potently suppress NK cells in vitro and in vivo in an MHV-3-induced mouse model of acute liver failure. This inhibitory effect is shown to require TGF-β and NO, and the interaction between ICAM-1and CDllb is required for recruitment of MDSCs and NK cells to the liver.. Therefore the purposes of this study are as the follows:1. To establish a mouse model of FHF by peritoneal injection with100PFU MHV-3.①To analyze the distribution of MDSCs in mouse bone marrow, peripheral blood, liver, spleen in the progression of MHV-3induced FHF.②To study the subgroups of heptic MDSCs in the progression of MHV-3induced FHF.③To investigate the role of LY6Ghi MDSCs to suppress natural killer cells④To investigate the role of LY6Chi MDSCs to activate natural killer cells2. Research the mechanisms of NK cells and MDSC cells accumulation in the liver Observed ICAM-1expression on hepatocytes and CDllb expression on NK cells and MDSCs at0and48h post MHV-3infaction.[METHODS]1.A mouse model of FHF was established by peritoneal infection with100PFU MHV-3.2. The proportion and number of MDSC cells in liver, spleen, blood and bone marrow at0,24,48and72h post MHV-3infection was analyzed by flow cytometry.3. The subgroups of MDSCs in liver at0,24,48,72h post MHV-3infection was analyzed by flow cytometry. The character of MDSCs subgroups was also analyzed by flow cytometry.4. In vitro,co-culture conditions NKcell and LY6Ghi MDSCs, The lever of IFN-y, produced, NKG2D expression,andYAC-l detected by hepatic NK cells, was also detected by intracellular cytokine staining or analyzed by flow cytometry.5. In vivo,Adoptive cell LY6Ghi MDSCs or antibody blocking MDSCs,analyzed NKG2D expression by flow cytometry. 6. In vitro,co-culture conditions NKcell and LY6Cint-hi LY6Gneg MDSCs, The lever of IFN-y, produced, NKG2D expression,andYAC-l detected by hepatic NK cells, was also detected by intracellular cytokine staining or analyzed by flow cytometry.The RAE-1expression by hepatic MDSCs was also analyzed at0,48h post MHV-3infection by flow cytometry.7. The ICAM-1expression on hepatic cell,The CDllb expression on MDSCs and NK cell were both analyzed at0,48h post MHV-3infection by flow cytometry.8. In vivo, antibody blocking ICAM-1, observed for fulminant hepatic failure model of survival curves.[RESULTS]1.Three subgroups of liver MDSCs are identified post MHV-3infection Normal livers have two MDSCs subpopulations:Ly6Chi Ly6GnegMDSCs and Ly6Cint Ly6Gneg MDSCs Livers of48hours post infection consisted of three different subpopulations:Ly6Chi Ly6GnegMDSCs, Ly6Cint Ly6Gneg MDSCs and Ly6Cint Ly6GnegMDSCs. Ly6Cint Ly6Gneg MDSCs with an SSChigh profile and ring-shaped nuclei. Ly6Chi Ly6GnegMDSCs with an SSCint profile and Ly6Cint Ly6Gneg MDSCs with an SSClow profile. Ly6Chi Ly6GnegMDSCs and Ly6Cint Ly6Gneg MDSCs all shape lymphocyte-like morphology.2.MDSCs have different homodynamic patterns in lymphoid organs. We observed subgroups of MDSCs subpopulations in lymphoid organs of healthy and infected mice. Fresh isolated lymphocytes stained with mAb against Gr-1and CD11b. For further characteristic, we analyzed Ly6C and Ly6G expression on CD11b positive population. Significantly higher percentages of CDllb+Gr-l+cells and Ly6GhiMDSCs accumulated in the blood post infection similar to liver). However, the percentage of these two populations decreased in liver,spleen and bone marrow It suggests that different homodynamic of MDSCs in different lymph organs may contribute to MDSCs accumulation in liver.3.Ly6Ghi MDSCs inhibits NK Cells in a TGF-β and NO dependent manner. In vitro, Ly6GhiMDSC staining were isolated from infected mice at48hours post infection, and cocultured at1:1ratio with NK cells from naive mice for12hours. LY6GhiMDSCs were suppressive and they suppressed NKG2D (MFI)expression, IFN-y production and cytotoxcity to YAC-I cells. The down-regulation of NKG2D expression by MDSCs remained in the transwell system, suggesting that MDSC-mediated inhibition of NK cells was not due to cell-to-cell interactions. Specific inhibitors of NO synthases arginase (L-NG-monomethyl arginine citrate, L-NMMA), TGF-β, and interleukin (IL)10were used. Neutralization of TGF-β or NO separately in the coculture system could restore NKG2D expression on NK cells. It suggests that TGF-β and NO were responsible for the suppression of NKG2D on NK cells by LY6G+MDSCs.4.DepIetion of MDSCs increase mortality and adoptive transfer of Ly-6Ghi cells inhibite NKG2D expression on NK Cells We chose two independent approaches. First, we depleted Grl+cells at24hours post infection using a Grl monoclonal antibody. This strategy shorted survival time compared to control isotype antibody. Secondly, we adoptively transfered two doses of GR1+or Ly6Ghi cells from mice48hours post infection to mice at beginning and24hours post infection. The transfer strategies did not increase survival rate. This maybe explained because of aggressively disease progress in this ALF mouse model. It is hard to observe the extension of survival. To observe the suppressive role of Ly-6Ghi cells to NK cells in vivo. Purified hepatic MNCs, Ly6G hi cells and non-Ly-6GhiMNCs from48hours infected mice were adoptively transferred into nai’ ve recipients. After24hours, NKG2D expression on hepatic NK cells was tested. Compared to MNCs and non-Ly-6Ghigroups which increased NKG2D expression, Ly-6Ghicells significantly decrease NKG2D expression. Adoptive transfer cells from infected mice took MHV-3into naive mice; this could explain the NK cells activation in MNCs and non-Ly-6Ghigroups. It suggest the suppressive role of Ly-6Ghicells in vivo.5.Ly6Cint-hi Ly6Gneg MDSCs activate NK Cells in RAE-1dependent manner. In vitro, Ly6Cint-hi Ly6Gneg MDSCs staining were isolated from infected mice at48hours post infection, and cocultured at1:1ratio with NK cells from naive mice for12hours. Ly6Cint-hi Ly6Gneg MDSCs increased NKG2D expression on NK cells. This may be partially explained by Ly6Cint-hiLy6Gneg MDSCs highly expressed RAE-1post infection. MDSCs-mediated down-regulation of NKG2D expression might be one of important mechanisms for the negative regulation of NK cells.6. MDSCs and NK cells accumulation in liver is in partly due to CDllb and ICAM-I interaction To further investigate NK cells and MDSCs accumulation in liver, we observed ICAM-1expression on hepatocytes and CDllb expression on NK cells and MDSCs. The level of ICAM-1expression on hepatocytes increased at48hours post infection. The percentage of CDllb positive NK cells significantly increased at48hours post infection, while MFI of CDllb on NK cells did not MFI of CDllb on Ly6Ghi MDSCs increased. Moreover, administration of anti-ICAM-1antibody into MHV-3infected mouse at24hours post infection resulted in a prolonged survival time and an improvement of histological manifestation. These data suggested ICAM-1interaction with CD11b is needed for MDSCs and NK cells crosstalk in liver.[CONCLUSION](1)An important aspect of our work is that we firstly demonstrated the hepatic Ly6Ghi MDSCs suppress NK cells activity in virus induced ALF. A model detailing the cross-talk between MDSCs and NK cells following MHV-3infection.(2)Our data demonstrated that three subpopulations within hepatic MDSCs can be distinguished by differential expression of Ly6G and Ly6C post MHV-3infection. Furthermore, the expression levels of Ly6C and Ly6G on MDSCs subsets are dynamically increased during the infection. Although the exact mechanism for this up regulation is unknown, this has major consequence for future research to define the phenotype of MDSCs in virus infection. Our data suggests the importance of including kinetic MDSCs phenotype analysis during acute virus infection. (3) We found that Ly6Ghi MDSCs efficiently inhibit NKG2D expression on NK cells in vitro and in vzvo.In our study, we demonstrated that cell-cell contact was unnecessary for Ly6Ghi MDSCs suppressing NK cells and that blockade of TGF-β or NO can reverse the suppression of NKG2D expression in vitro.(4) We found that ICAM-1is expressed on hepatocytes and to be rapidly upregulated following MHV-3infection. Our data indicated that the interaction between integrin αm (CD11b), which is highly expressed on Ly6Ghi MDSCs and NK cells post infection, and ICAM-1may facilitate the interaction of NK cells and MDSCs in liver.