LPS Induces Fulminant Hepatitis and Mortality in NS5A Tg Mice in a TLR4-Dependent Manner.

To test the functionality of NS5A-induced TLR4, we created 4 genetic mouse lines with the identical background C57BL/6: (i) WT mice (WT); (ii) WT mice with TLR4 deficiency (Tlr4^−/−); (iii) NS5A Tg mice (NS5A); and (iv) NS5A Tg mice with TLR4 deficiency (Tlr4^−/−NS5A). These mice were challenged by intraperitoneal injection of an acute sublethal dose of LPS (25 mg/kg). This challenge expectedly and markedly raised the serum levels of aspartate aminotransferase (AST) and TNF-α in WT, whereas these parameters were significantly attenuated in Tlr4^−/− (Fig. 1 D and E). LPS-challenged WT mice did not show conspicuous liver pathology (Fig. 1C Top) or mortality [supporting information (SI) Fig. S1A]. In contrast, in NS5A Tg mice, LPS caused 30% mortality (Fig. S1A), fulminant hepatitis characterized by massive hemorrhagic liver necrosis and inflammation (Fig. 1C Middle), and augmented elevations of serum AST and TNF-α (Fig. 1 D and E). Importantly, all of these changes were largely abrogated in Trl4^−/−NS5A mice (Fig. 1 C–E and Fig. S1A), supporting the role of TLR4 in LPS-induced hepatitis and mortality in NS5A mice. Next, we validated whether NS5A expression augmented LPS-induced signal transduction via TLR4 by assessing the interaction of TGF-α-activated kinase 1 (TAK1) with TNFR-associated factor 6 (TRAF6) or IL-1 receptor-associated kinase-1 (IRAK1), the signaling events immediately downstream of LPS-TLR4-CD14 binding (13). Coimmunoprecipitation analysis of liver protein extracts readily detected enhanced TAK1–TRAF6 and TAK1–IRAK1 interactions in NS5A mice given LPS, whereas these protein–protein interactions were not observed in WT or Tlr4^−/−NS5A mice under the same immunoblotting condition (Fig. 1F). We also analyzed phosphorylation of JNK and IκBα, 2 further downstream parameters of TLR4 signaling. As shown in Fig. 1G, phosphorylation of JNK and IκBα (p-JNK, p- IκBα) was also prominent in NS5A mice but not in WT or Tlr4^−/−NS5A mice (Fig. 1G). These results demonstrate augmented activation of TLR4 signaling in the liver of LPS-challenged NS5A mice and their sensitization for LPS-induced fulminant hepatitis.

Because TLR4 was primarily induced in hepatocytes by hepatocyte-specific expression of NS5A, the accentuated liver damage in NS5A mice is presumed to have resulted from heightened TLR4 activation in the parenchymal cells. However, Kupffer cells still represent another obvious cellular site of TLR4 signaling. Thus, to determine whether or to what extent Kupffer cells contribute to liver injury induced by LPS in NS5A mice, we depleted Kupffer cells by administration of liposome-encapsulated Clodronate (27) 2 days before LPS injection. This method depletes more than 95% of Kupffer cells (27) and largely (75≈80%) prevented LPS-induced increases in serum AST and TNF-α levels in WT, confirming that Kupffer cells are the predominant site of TLR4 activation. In NS5A, however, the same manipulation reduced these parameters only by 35% and 39%, respectively (Fig. S1 B and C) and did not attenuate the mortality rate (data not shown). From these results, we conclude that Kupffer cell-derived TNF-α accounted for ≈40% of total TNF-α produced in response to LPS in NA5A mice and had a only minor if no role in liver damage and mortality in this model. Conversely, these results suggest that the remaining 60% of TNF-α in the serum of these mice was produced by TLR4-expressing hepatocytes.

Expression of NS5A and TLR4 in Hepatitis C Patient Livers.

Next, we assessed the clinical relevance of our finding on TLR4 induction in NS5A mice. Liver protein extracts were obtained from healthy subjects, patients with HCV infection, and HCV patients with a history of alcoholism. These samples and the samples from NS5A mouse livers were concomitantly analyzed for the expression of NS5A by immunoblot analysis. As shown in Fig. S2A, the expression of NS5A is increased in HCV patients with or without alcoholism compared with healthy subjects. A densitometric analysis of normalized NS5A expression shows that the expression in the patient livers is about a third of the level seen in NS5A mice (Fig. S2B). These results indicate that NS5A Tg expression in our mouse model is not extremely unphysiological. Next, we performed immunocytochemistry on liver cryosections from HCV patients with or without alcoholism to assess the expression of TLR4 and 2 downstream parameters of TLR4 signaling: p-JNK and p-IκBα. In these samples, increased colocalization of TLR4 with p-JNK and p-IκBα was noted, particularly in HCV patients with alcoholism (Fig. S2 D and E). These results support the clinical relevance of our finding on TLR4 induction and activation in NS5A mice.

NS5A-Induced TLR4 Aggravates Alcoholic Steatohepatitis.

Because NS5A mice are sensitive to LPS due to TLR4 induction, and alcohol-induced liver injury is known to be mediated by endotoxin, we tested whether NS5A Tg mice are more susceptible to alcohol-induced liver damage. For this, the same 4 genetic lines of mice described above (WT, Tlr4^−/−, NS5A, and Tlr4^−/−NS5A) were fed either control or ethanol diet by intragastric infusion that allowed maximal ethanol intake for 4 weeks (see experimental design in Fig. S3A). Ethanol feeding in WT mice resulted in diffuse (2+≈4+) fatty liver with or without small foci of inflammation (Table S1) and a 2.7-fold increase in plasma alanine aminotransferase (ALT) levels (95 ± 18 units/L) compared with control diet-fed WT mice (Fig. S3B). In contrast, ethanol-fed NS5A Tg mice displayed an additional 2-fold increment in ALT elevation (181 ± 21 units/L) compared with ethanol-fed WT mice (Fig. S3B). Spotty and submassive liver necrosis, as well as infiltration of mononuclear cells, neutrophils, and eosinophils in the necrotic midzone region, was also observed in these mice (Fig. S3G). This pathology resembles coagulative necrosis commonly observed in chronically ethanol-fed rodents given acute LPS and confirmed that NS5A Tg mice are more susceptible to alcoholic steatohepatitis. This pathology and the elevation of the plasma ALT level were largely abolished in Tlr4^−/−NS5A mice (Table S1 and Fig. S3 D and H), confirming the pathogenic role of TLR4 in this mouse model. It should be noted that the plasma endotoxin levels in NS5A Tg and WT mice were equally elevated by alcohol feeding compared with pair-fed control animals (Fig. S3C).

Next, we determined the role of endotoxin in alcoholic steatohepatitis in NS5A Tg mice by intragastric administration of polymyxin B (150 mg/kg per day) and neomycin (450 mg/kg per day) for 4 days before alcohol feeding and during the entire feeding period. This antibiotic treatment reduced ALT levels (Fig. S3E) and liver pathology in ethanol-fed NS5A Tg mice (Fig. S3I and Table S1). Conversely, LPS was administered weekly via the intragastric tube to ethanol-fed NS5A Tg mice to test whether this manipulation accentuates alcoholic liver damage. As expected, LPS aggravated liver damage caused by alcohol (Table S1) and led to 2.5-fold higher serum ALT levels compared with alcohol-fed NS5A mice without LPS (Fig. S3E). These results indicate the importance of endotoxin-activated TLR4 signaling in the pathogenesis of aggravated steatohepatitis in alcohol-fed NS5A Tg mice. Oxidant damage is a key feature of alcoholic liver damage that can be further potentiated by endotoxin (28, 29). Thus, we measured the hepatic content lipid peroxides in WT and NS5A Tg mice fed alcohol or control diet. Alcohol feeding increased the lipid peroxide levels 2-fold in WT mice. In NS5A Tg mice, this effect was significantly accentuated with a 3.3-fold elevation, indicating enhanced oxidative damage in alcohol-fed NS5A Tg mice (Fig. S3J).

NS5A-Induced TLR4 Causes Synergistic Liver Oncogenesis by Long-Term Alcohol Feeding.

We next extended our study to determine whether NS5A-mediated TLR4 induction causes liver tumor after prolonged alcohol feeding. For this experiment, we fed the same 4 genetic lines of mice Lieber-DeCarli diet containing 3.5% (wt/vol) ethanol or isocaloric dextrin for 12 months. This liquid diet containing the lower ethanol concentration was fed ad libitum, and this regimen alleviated the high mortality commonly associated with this type of long-term feeding. Total daily caloric intakes from the control or ethanol diet by different genetic groups were not significantly different (WT: 17.2 ± 3.4 vs. 15.3 ± 2.8, Tlr4^−/−: 18.1 ± 3.1 vs. 16.1 ± 4.4, NS5A: 18.1 ± 4.1 vs. 15.7 ± 3.0, and Tlr4^−/−NS5A: 17.6 ± 3.6 vs. 16.3 ± 4.0 mL/mouse per day). Liver tumor was not detected in WT, NS5A Tg, or Tlr4^−/−NS5A mice fed the control diet or WT mice fed the alcohol diet. In contrast, liver tumor was found in 23% of ethanol-fed NS5A Tg mice (Fig. 2 and Table S2) but was completely absent in Tlr4^−/−NS5A Tg mice (Table S2). Most tumors detected in NS5A mice were hepatoma (Fig. 2). WT and NS5A mice studied for this experiment had relatively even sex distributions (49% and 55% females vs. 51% and 45% males), and the tumor incidence was not statistically different between the 2 sexes (Table S3). Alcohol feeding increased serum TNF-α levels 4-fold in WT mice and 6-fold in NS5A Tg mice, and this difference was significant (Table S2). TLR4 deficiency significantly and largely attenuated this increment in alcohol-fed NS5A mice (Table S2). Enhanced TLR4 signaling by NS5A and alcohol feeding was validated by increased TAK1 interaction with TRAF6 and by elevated p-JNK and p-IκB in the livers of alcohol-fed NS5A mice but not alcohol-fed WT or Tlr4^−/−NS5A mice (Fig. S4 A and B). Colocalization of TLR4 with TNF-α, p-IκB, and p-JNK was also evident in liver sections of alcohol-fed NS5A but not WT mice (Fig. S4 C and D). These data demonstrate that alcohol and NS5A synergistically induce liver tumors through enhanced expression of TLR4 and signaling.

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Fig. 2.

HCV NS5A induced liver tumors after 12-month alcohol feeding, as shown in this representative photograph (Left) of gross liver appearance and a microphotograph (Right) of an H&E-stained section of the tumor. (Magnification: 100×).

Nanog as a TLR4 Downstream Gene Induced by NS5A and Alcohol.

To understand the molecular mechanisms of the synergism demonstrated in alcohol-fed NS5A mice, we performed microarray analysis on RNA samples extracted from non-tumor-bearing portions of liver tissues from NS5A Tg and WT mice fed alcohol as described in detail in SI Methods. Of more than 39,000 transcripts and variants examined by using more than 45,000 probe sets, 83 transcripts showed increased expression with a cutoff value of 4.0-fold (balanced differential expression) in NS5A mouse livers. The lists of differentially regulated genes are shown in Fig. S5. Of note are induction of a stem cell marker (i.e., Nanog), TLR4 downstream cytokines (e.g., IFN-α4), and apoptosis-related genes (e.g., Bcl2a1a and Bcl11a) as well as down-regulation of the epigenetic transcriptional regulator trithorax group of proteins (e.g., ASH1 and ASH2) and developmental transcription factors (e.g., Hox, Myo, and Fox families).

Induction of the stem cell marker Nanog in alcohol-fed NS5A mice was intriguing in light of the report that a particularly poor prognostic subtype of human HCC is derived from hepatic progenitor cells (30). This induction was confirmed by immunofluorescence microscopy (Fig. 3A), immunoblotting (Fig. 3B), and quantitative RT-PCR (Fig. 3C). Nanog was colocalized with another stem cell marker, CD133 or CD49f, in liver tumors of NS5A Tg mice fed alcohol (Fig. 3A), suggesting the presence of cancer stem cells. Nanog induction was dependent on NS5A and alcohol (Fig. 3 B and C) and abrogated in Tlr4^−/−NS5A mice fed alcohol (Fig. 3C). We next tested the direct relationship of Nanog induction with TLR4 activation by analyzing Nanog expression in LPS-treated Huh7 cells transduced with an NS5A or control vector with or without TLR4 knockdown with short-hairpin RNA (shRNA). As shown in Fig. 3D, LPS up-regulated Nanog expression in NS5A-transduced cells but not in control vector-transfected cells. This induction of Nanog by LPS was significantly reduced by shRNA directed against TLR4 but not by scrambled shRNA (Fig. 3D). To assess the efficiency of TLR4 knockdown, we performed immunoblot analysis. As shown in Fig. 3E, induction of TLR4 protein by NS5A was decreased approximately by 80% with TLR4 shRNA. Overexpression of Tlr4 (TLR4+) but not the mutant with deletion of the cytoplasmic domain (TLR4ΔCyt) in Huh7 cells also increased the expression of Nanog in the presence of LPS, further validating that activated TLR4 enhances Nanog expression (Fig. 3D). To determine whether NS5A induces Nanog expression via TLR4 at the transcriptional level, we performed a reporter assay using a Nanog promoter. LPS treatment increased the reporter activity in the cells transduced with NS5A. This induction was attenuated with TLR4 shRNA but not with control shRNA (Fig. 3F). Transduction of wild-type TLR4 but not the mutant TLR4 also caused the promoter activation in the presence of LPS. These results demonstrate that the promoter of the Nanog gene is directly activated by TLR4, and Nanog is a novel downstream gene of TLR4 signaling.

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Fig. 3.

(A) Confocal immunofluorescent microscopy demonstrates colocalization of the stem cell marker Nanog with CD133 or CD49f in the tumor-bearing tissue from NS5A mice fed alcohol for 12 months. (Magnification: 100×.) (B) Nanog protein was induced in the livers of NS5A mice fed alcohol, as determined by immunoblot analysis. Immunoblotting of β-actin served as a loading control. (C) Nanog mRNA was induced in the livers of NS5A mice fed ethanol, as determined by real-time PCR (*, P < 0.03 compared with ethanol-fed WT mice). This induction was attenuated in ethanol-fed Tlr4^−/−NS5A mice (*, P < 0.03 compared with ethanol-fed NS5A). (D) LPS induced Nanog mRNA in Huh7 cells transduced with an NS5A expression vector (*, P < 0.002 compared with the cells transduced with the empty vector and control shRNA). This induction was suppressed by retroviral expression of shRNA for TLR4 (**, P < 0.05). Nanog mRNA also was induced by LPS treatment in the cells transduced with a retroviral vector expressing wild-type TLR4 (TLR4+, *, P < 0.05 compared with the control cells), but not in the cells transduced with the mutant TLR4 vector (TLR4ΔCyp). (E) Efficient knockdown of TLR4 with the retroviral shRNA vector is evident in this immunoblot analysis and densitometric analysis revealing an 82% reduction (*, P < 0.05). (F) LPS added to the culture activated the Nanog promoter in Huh7 cells transfected with the promoter-luciferase construct (*, P < 0.002 compared with the control cells). TLR4 knockdown with shRNA as above abrogated the promoter activation (**, P < 0.05). Expression of wild-type TLR4 (TLR4+, *, P < 0.05) but not mutant TLR4 (TLR4ΔCyp) conferred the cells LPS-induced Nanog promoter activation. Relative light unit values were normalized by the activity of the Renilla luciferase construct driven by SV40 promoter as a transfection control.

We thank Qing-gao Deng, Jiaohong Wang, Sean Vorah, and Jeffrey Huang (University of Southern California) for mouse experiments; Michael Karin (University of California at San Diego) for discussions; Paul Robson (Genome Institute of Singapore, Immunos, Singapore) for Nanog-promoter luciferase construct; Steven Weinman (University of Texas) for suggestions; and Moli Chen and Alex Trana for histological service. The study was supported by pilot project funding; the Animal and Morphology Cores of the Southern California Research Center for ALPD & Cirrhosis (P50 AA011999) funded by the National Institute on Alcohol Abuse and Alcoholism/National Institutes of Health (NIAAA/NIH); NIH Grants AI 40038, CA123328, and CA108302; and the Medical Research Service of the Veterans Administration.