Natural Killer Cells Ameliorate Liver Fibrosis by Killing Activated Stellate Cells in NKG2D-Dependent and Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Dependent Manners

Background & Aims: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure. Methods: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon γ. Results: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon γ gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon γ enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor–related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor–related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid–activated natural killer cells against activated hepatic stellate cells. Conclusions: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor–related apoptosis-inducing ligand–dependent mechanisms, thereby ameliorating liver fibrosis. Background & Aims: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure. Methods: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon γ. Results: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon γ gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon γ enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor–related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor–related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid–activated natural killer cells against activated hepatic stellate cells. Conclusions: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor–related apoptosis-inducing ligand–dependent mechanisms, thereby ameliorating liver fibrosis. Viral hepatitis, which affects half a billion people worldwide, is a major cause of liver injury, fibrosis, and cirrhosis; however, the cellular and molecular mechanisms underlying the progression of liver disease during infection are not fully understood.1Liang T.J. Rehermann B. Seeff L.B. Hoofnagle J.H. Pathogenesis, natural history, treatment, and prevention of hepatitis C.Ann Intern Med. 2000; 132: 296-305Crossref PubMed Scopus (793) Google Scholar, 2Fung S.K. Lok A.S. 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Lymphocyte activation via NKG2D towards a new paradigm in immune recognition?.Curr Opin Immunol. 2002; 14: 306-311Crossref PubMed Scopus (190) Google Scholar, 26Ortaldo J.R. Young H.A. Mouse Ly49 NK receptors balancing activation and inhibition.Mol Immunol. 2005; 42: 445-450Crossref PubMed Scopus (37) Google Scholar These inhibitory receptors include inhibitory killer cell immunoglobulin-like receptor KIR, Ly-49A, and CD94/NKG2 receptors. Among the several stimulatory NK cell receptors, the NKG2D receptor is the best defined and is expressed on both human and mouse NK cells, where it is recognized by several ligands, including MHC class I chain–related gene A and UL16-binding protein for human NK cells and retinoic acid early inducible 1 (RAE1), histocompatibility 60, and mouse UL16-binding protein-like transcript 1 for mouse NK cells.22Lanier L.L. NK cell recognition.Annu Rev Immunol. 2005; 23: 225-274Crossref PubMed Scopus (2233) Google Scholar, 23Lanier L.L. Natural killer cell receptor signaling.Curr Opin Immunol. 2003; 15: 308-314Crossref PubMed Scopus (295) Google Scholar, 24Vivier E. Nunes J.A. Vely F. Natural killer cell signaling pathways.Science. 2004; 306: 1517-1519Crossref PubMed Scopus (496) Google Scholar, 25Vivier E. Tomasello E. Paul P. Lymphocyte activation via NKG2D towards a new paradigm in immune recognition?.Curr Opin Immunol. 2002; 14: 306-311Crossref PubMed Scopus (190) Google Scholar, 26Ortaldo J.R. Young H.A. Mouse Ly49 NK receptors balancing activation and inhibition.Mol Immunol. 2005; 42: 445-450Crossref PubMed Scopus (37) Google Scholar It has been shown that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)+ liver NK cells express low levels of the NK inhibitory receptor Ly-49A, which could be an important mechanism contributing to the cytotoxicity of liver NK cells against self hepatocytes28Ochi M. Ohdan H. Mitsuta H. Onoe T. Tokita D. Hara H. Ishiyama K. Zhou W. Tanaka Y. Asahara T. Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice.Hepatology. 2004; 39: 1321-1331Crossref PubMed Scopus (116) Google Scholar and tumors.29Takeda K. Hayakawa Y. Smyth M.J. Kayagaki N. Yamaguchi N. Kakuta S. Iwakura Y. Yagita H. Okumura K. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells.Nat Med. 2001; 7: 94-100Crossref PubMed Scopus (588) Google Scholar Here we show that NK cells kill activated HSCs, but not quiescent HSCs, which is likely mediated via an NKG2D/RAE1-dependent mechanism because expression of RAE1, the NKG2D ligand, was detected at high levels on activated HSCs but not on quiescent HSCs. Moreover, the cytotoxicity of NK cells against activated HSCs seems to be dependent on TRAIL also.Materials and MethodsMaterialsPoly I:C, Gey’s balanced salt solution, and OptiPrep were purchased from Sigma (St Louis, MO). Pronase E and collagenase D were obtained from Roche (Indianapolis, IN).MiceEight- to 10-week old male C57BL/6J, IFN-γ−/− mice (C57BL/6J background), perforin−/− mice (C57BL/6J background), and Fas ligand (FasL)−/− mice (C57BL/6Smn background) were purchased from the Jackson Laboratory (Bar Harbor, ME). CD1d−/− mice (natural killer T cell [NKT] deficient) on a BALB/c background were originally purchased from the Jackson Laboratory and backcrossed with C57BL/6J mice for at least 8 generations. All mice used in this study were housed in a specific pathogen–free facility and were cared for in accordance with National Institutes of Health guidelines.Liver Injury and Fibrosis Induced by a 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine Diet and Carbon TetrachlorideFor 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet–induced liver injury, mice were fed a diet containing DDC (0.1% wt/wt in Purina 5015 mouse chow; Bio-Serv, Frenchtown, NJ) for 1–4 weeks. In the carbon tetrachloride–induced liver injury model, mice were injected intraperitoneally (IP) with carbon tetrachloride (10% in olive oil, 2 mL/kg, 3 times a week) for 2–3 weeks. After mice were killed, liver tissues were frozen in liquid nitrogen or fixed in 10% buffered formalin and embedded in paraffin.Depletion of Natural Killer Cells by Anti–Asialo GM-1 AntibodiesTo deplete NK cells (NK1.1+CD3−), anti-asialo GM-1 (ASGM-1) antibody (Ab; 100 μL per mouse; catalog no. 986-10001; Wako, Richmond, VA) was injected IP into mice. After 24 hours, depletion of NK (NK1.1+CD3−) cells was confirmed by flow cytometry.11Sun R. Gao B. Negative regulation of liver regeneration by innate immunity (natural killer cells/interferon-gamma).Gastroenterology. 2004; 127: 1525-1539Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar To chronically deplete NK cells, mice were treated with anti–ASGM-1 every 48 hours for 2 weeks.Treatment of Mice With Polyinosinic-Polycytidylic AcidWe have previously shown that injection of mice with poly I:C markedly induces accumulation and activation of NK cells in the liver, with the peak effect occurring at 24 hours and returning to basal levels at 48 hours after injection.30Dong Z. Wei H. Sun R. Hu Z. Gao B. Tian Z. Involvement of natural killer cells in polyI:C-induced liver injury.J Hepatol. 2004; 41: 966-973Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Therefore, for chronic treatment with poly I:C, mice were injected IP with poly I:C every 48 hours for 1–2 weeks. For mice in the DDC diet and poly I:C cotreatment group, mice were injected with poly I:C 24 hours before DDC feeding and then maintained on the DDC diet with poly I:C injected every 48 hours for 1–2 weeks. For mice in the carbon tetrachloride and poly I:C cotreatment group, mice were injected IP with carbon tetrachloride (10% in olive oil, 2 mL/kg, 3 times a week) for 1 week and then treated with carbon tetrachloride (IP 3 times a week) and poly I:C (IP every 48 hours) for an additional 1–2 weeks. Control mice received carbon tetrachloride treatment plus saline injection. For acute poly I:C treatment, mice were injected IP with poly I:C once. After 16 hours, liver mononuclear cells (MNCs) or NK cells were isolated and used as effector cells in cytotoxicity assays or for reverse-transcription polymerase chain reaction (RT-PCR) analyses. The dose of poly I:C used in this study was 1 μg/g unless specified otherwise.Histology and ImmunohistochemistryAfter routine processing, liver sections 5 μm thick were stained with H&E for histological analysis or Masson trichrome for collagen deposition. Immunostaining for α-SMA was performed on formalin-fixed, paraffin-embedded sections by using monoclonal α-SMA primary Ab (clone 1A4; Dako, Carpinteria, CA) and a biotinylated alkaline phosphatase–conjugated secondary Ab. Fast Red (Dako) was used as the chromagen/substrate. The collagen area/portal vein area and α-SMA–positive area/portal vein area were quantified by digital imaging with National Institutes of Health Scion Image and Adobe Photoshop (San Jose, CA) by using Masson trichrome and α-SMA–stained sections, respectively.Double Staining With Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridine Triphosphate Nick-End Labeling and α-Smooth Muscle Actin AntibodyHSC apoptosis in the liver was determined by double staining with terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and α-SMA Ab, as described previously.31Iredale J.P. Benyon R.C. Pickering J. McCullen M. Northrop M. Pawley S. Hovell C. Arthur M.J. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (930) Google Scholar Apoptotic cells in sections of mouse livers were detected by using the In Situ Cell Death Detection Kit, POD (Roche, Indianapolis, IN), according to the manufacturer’s instructions. TUNEL-positive nuclei were identified by the AEC Substrate Kit for Peroxidase from Vector Laboratories (Burlingame, CA) as red staining. Then, immunostaining for α-SMA with an alkaline phosphatase–conjugated secondary Ab was detected by a Vector Blue Alkaline Phosphatase Kit III. The number of TUNEL-positive (red) and α-SMA (blue) cells per field were counted.Western BlottingWestern blotting was performed as described previously.32Radaeva S. Jaruga B. Hong F. Kim W.H. Fan S. Cai H. Strom S. Liu Y. El-Assal O. Gao B. Interferon-alpha activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes.Gastroenterology. 2002; 122: 1020-1034Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar Protein bands were visualized by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ).Reverse-Transcription Polymerase Chain ReactionThe RT-PCR was performed as described previously,32Radaeva S. Jaruga B. Hong F. Kim W.H. Fan S. Cai H. Strom S. Liu Y. El-Assal O. Gao B. Interferon-alpha activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes.Gastroenterology. 2002; 122: 1020-1034Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar and the primers used are listed in Table 1.Table 1RT-PCR Primers Used in This StudyGenesForward (5′–3′)Reverse (5′–3′)PCR (base pairs)NKG2DGCA TTG ATT CGT GAT CGA AAGCC ACA GTA GCC CTC TCT TG374Ly-49ATTC TGG AAT CCC TCA ACA GGGAA GGA ACC ACG AGC TGA AG247RAE1αAGG TTG TGA GCT GCT CGA TTGGG GTA GGA TCC TTG ATG GT272RAE1βGCT GCA GTT CAA GAC ACC AAGGG GTA GGA TCC TTG ATG GT246RAE1γATT TGC ATT TGC GAT GTG AAGCT GTA TTG CCT GGC ATT TT382RAE1δGCT GCA GTT CAA GCA ACC AACCA CGA AGC ACT TCA CTT CA272RAE1εGCT GCA GTT CAA GAC ACC AATCC ACT GAG CAC TTC ACG TC285H-60TGT GCT GAT TTG TCC CAA AGAAT TTG TTG AGG CAG CGA CT299Mult1CAT GCC ATT GGT GCT CAT AGTGC TTG TGT CAA CAC GGA AT250H2-D1GGA AAA GGA GGG GAC TAT GCGGA CAG GGT CCT GGT GTA GA273H2-D4CCA GTG GAT GTA TGG CTG TGTCT CCT TCC CTC CTG AGA CA444Clec2DCTC GGT TTG ACA ACC AGG ATGAT CCC GTT GTT GTT CAG GT212PerforinGAT GTG AAC CCT AGG CCA GAGGT TTT TGT ACC AGG CGA GA155TRAILCCC TGC TTG CAG GTT AAG AGGGC CTA AGG TCT TTC CAT CC220HGFTTC CCA GCT GGT CTA TGG TCTGG TGC TGA CTG CAT TTC TC237TGF-β1TTG CTT CAG CTC CAC AGA GATGG TTG TAG AGG GCA AGG AC183PDGFαGAG ATA CCC CGG GAG TTG ATAAA TGA CCG TCC TGG TCT TG238PDGFβCCT CGG CCT GTG ACT AGA AGGGA CGA GGG GAA CAA CAT TA267TGF-αCTG AAG GGA AGG ACT GCT TGGTC CAC TGG CCT CTT CTC TG272FGF-2AGC GGC TCT ACT GCA AGA ACTCG TTT CAG TGC CAC ATA CC299IFN-γACT GGC AAA AGG ATG GTG ACTGA GCT CAT TGA ATG CTT GG237Fas LTTG TTT TGG GGA GGA GTC AGGGG GTA GGT GCT CAG TGT GT175JECCC AAT GAG TAG GCT GGA GAGAA CTG CCT TTG CCT TCT TG199MIP-1βCCC ACT TCC TGC TGT TTC TCGAG GAG GCC TCT CCT GAA GT236I-TACATG AAC GGC TGC GAC AAA GTGCA TGT TCC AAG ACA GCA GA221α-SMACTG ACA GAG GCA CCA CTG AAGAA GGA ATA GCC ACG CTC AG288TLR3TTG TCT TCT GCA CGA ACC TGCGC AAC GCA AGG ATT TTA TT204β-actinAGC CAT GTA CGT AGC CAT CCCTC TCA GCT GTG GTG GTG AA227Mult1, mouse UL16-binding protein-like transcript 1; H-60, histocompatibility 60; HGF, hepatocyte growth factor; FGF, fibroblast growth factor; JE, monocyte chemoattractant protein-I; I-TAC, IFN-γ–inducible T-cell α chemoattractant. Open table in a new tab Isolation and Culture of Hepatic Stellate CellsHSCs were isolated from murine livers through sequential digestion with collagenase D (1 mg/mL) and pronase E (2 mg/mL) in Gey’s balanced salt solution followed by discontinuous density centrifugation in 11.5% OptiPrep as described previously.33Arthur M.J. Friedman S.L. Roll F.J. Bissell D.M. Lipocytes from normal rat liver release a neutral metalloproteinase that degrades basement membrane (type IV) collagen.J Clin Invest. 1989; 84: 1076-1085Crossref PubMed Scopus (155) Google Scholar, 34Oakley F. Meso M. Iredale J.P. Green K. Marek C.J. Zhou X. May M.J. Millward-Sadler H. Wright M.C. Mann D.A. Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis.Gastroenterology. 2005; 128: 108-120Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar By use of trypan blue staining, the viability of the cells was determined to be 98%. The purity of the cells was also assessed visually by light microscopy examination of typical lipid droplet appearance, by fluorescence microscopy examination of vitamin A autofluorescence, and by immunochemistry examination of desmin expression. Typically, the purity of stellate cells was more than 90%.Isolation of Mouse Liver Mononuclear Cells and Natural Killer (NK1.1+CD3−) CellsLiver MNCs were isolated as described previously.35Hong F. Jaruga B. Kim W.H. Radaeva S. El-Assal O.N. Tian Z. Nguyen V.A. Gao B. Opposing roles of STAT1 and STAT3 in T cell-mediated hepatitis regulation by SOCS.J Clin Invest. 2002; 110: 1503-1513Crossref PubMed Scopus (255) Google Scholar To purify liver NK cells (NK1.1+CD3−), CD3− liver MNCs were separated from liver MNCs by negative magnetic cell sorting according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). Subsequently, NK1.1+CD3− cells were purified by positive magnetic cell sorting by using anti-NK1.1 monoclonal Ab according to the manufacturer’s protocol (Miltenyi Biotec). Approximately 93% of the MACS-purified cells were NK1.1+CD3− cells.Flow Cytometric Analysis of Natural Killer Cells, TRAIL, and NKG2D ExpressionNK cells, T cells, NKG2D, Ly-49A, FasL, and TRAIL expression on MNCs from the livers of C57BL/6 mice and IFN-γ−/− mice were determined by using anti-NK1.1, anti-CD3, anti–Ly-49A, anti-FasL (BD PharMingen, San Diego, CA), and anti-TRAIL, anti-NKG2D Abs (eBioscience, San Diego, CA) by FACSCalibur (BD Biosciences, Mountain View, CA).Cell-Mediated CytotoxicityThe ToxiLight kit (Cambrex, Rockland, ME) was used to measure cell-mediated cytotoxicity against primary HSCs. Briefly, freshly isolated HSCs were plated onto round-bottom, 96-well plates. MNCs or NK cells were subsequently added as target cells at target–effector ratios ranging from 1:2 to 1:25. The release of adenylate kinase from damaged cells was measured from the culture medium by using the ToxiLight kit. To directly visualize the killing process and confirm whether MNCs or NK cells directly killed HSCs, the LIVE/DEAD Cell-Mediated Cytotoxicity Kit (Molecular Probes, Eugene, OR) was used. After target cells were loaded with a green fluorescent membrane stain, 3,3′-dioctadecyloxacarbocyanine, the plates were incubated with effector cells, and the coculture was stained with propidium iodide. The results showed that most propidium iodide–stained cells were also positive for green fluorescence, thus indicating that stellate cells were killed by either MNCs or NK cells.Statistical AnalysisData are expressed as means ± SEM. To compare values obtained from 3 or more groups, 1-factor analysis of variance was used, followed by the Tukey post hoc test. To compare values obtained from 2 groups, the Student t test was performed. Statistical significance was taken at the P < .05 level.ResultsPolyinosinic-Polycytidylic Acid Treatment Attenuates Liver Fibrosis Induced by DDC Diet or Carbon Tetrachloride InjectionFeeding mice with a diet containing DDC induced liver injury as evidenced by elevation of serum alanine aminotransferase levels and necrosis in the liver (Figure 1A). Fibrosis was also induced, as shown by collagen deposition (blue staining by Masson trichrome) and HSC activation (α-SMA–positive staining; Figure 1B and C). To investigate the effect of NK cell activation on liver fibrosis, mice were fed the DDC diet and cotreated with poly I:C, which activates NK cells and induces accumulation of NK cells in the liver but produces only very mild liver injury.30Dong Z. Wei H. Sun R. Hu Z. Gao B. Tian Z. Involvement of natural killer cells in polyI:C-induced liver injury.J Hepatol. 2004; 41: 966-973Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 36Twilley T.A. Mason L. Talmadge J.E. Wiltrout R.H. Increase in liver-associated natural killer activity by polyribonucleotides.Nat Immun Cell Growth Regul. 1987