Potential conflict of interest: Nothing to report.
Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) is required for liver fibrosis. This study investigates the role of NOX in ROS production and the differential contribution of NOX from bone marrow (BM)-derived and non–BM-derived liver cells. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine-choline-deficient (MCD) diet for 10 weeks in wild-type (WT) mice and mice deficient in p47phox (p47phox knockout [KO]), a component of NOX. The p47phox KO chimeric mice were generated by the combination of liposomal clodronate injection, irradiation, and BM transplantation of p47phox KO BM into WT recipients and vice versa. Upon BDL, chimeric mice with p47phox KO BM-derived cells, including Kupffer cells, and WT endogenous liver cells showed a ∼25% reduction of fibrosis, whereas chimeric mice with WT BM-derived cells and p47phox KO endogenous liver cells, including hepatic stellate cells, showed a ∼60% reduction of fibrosis. In addition, p47phox KO compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury, but a ∼50% reduction in fibrosis. Cultured WT and p47phox KO hepatocytes treated with free fatty acids had a similar increase in lipid accumulation. Free fatty acids promoted a 1.5-fold increase in ROS production both in p47phox KO and in WT hepatocytes. Conclusion: NOX in both BM-derived and non–BM-derived cells contributes to liver fibrosis. NOX does not play a role in experimental steatosis and the generation of ROS in hepatocytes, but exerts a key role in fibrosis. (HEPATOLOGY 2010;)
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Liver fibrosis is the consequence of chronic liver injury and represents an important cause of mortality worldwide.1, 2 Liver fibrosis results from inflammation associated with the production of chemokines and cytokines that stimulate and act on different cells in the liver, including hepatic stellate cells (HSCs), Kupffer cells (KCs), hepatocytes (HEPs), and endothelial cells.3, 4 The endpoint of advanced liver fibrosis is cirrhosis, in which the fibrous scar and regenerating nodules lead to hepatocellular dysfunction, portal hypertension, and hepatocellular carcinoma.5, 6 An inflammation–fibrosis–cancer axis has therefore been proposed.7
Oxidative stress plays an important role in inflammation, fibrosis, and cancer.8-10 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), a key source of reactive oxygen species (ROS) in the liver,11 may therefore represent an important new therapeutic target.12 NOX is a multiprotein complex that catalyzes ROS formation in response to many stimuli.9, 13 Macrophages, including KCs, express the phagocytic form of NOX that plays a crucial role in antimicrobial defense.8, 14-16 The nonphagocytic form of NOX is expressed in other cell types, such as vascular smooth muscle cells and cardiac myofibroblasts, and is required for the proper activation of many intracellular signalling pathways, including mitogen-activated protein kinase and phosphoinositide 3-kinase.11, 17, 18 We have previously shown that NOX is required for hepatic fibrosis in vivo.9 KCs express the majority of NOX in the liver and play an important role in HSC activation.19 However, NOX is also expressed by HSCs and is able to mediate HSC activation in response to fibrogenic agonists such as platelet-derived growth factor, angiotensin II, and leptin.9, 18, 20 Furthermore, HEPs contain NOX,21 providing a potentially additional sources of ROS in inflammation and fibrosis. However, the relative contribution of NOX in KCs, HEPs, and HSCs to hepatic inflammation and fibrosis remains unknown. This study demonstrates that NOX-mediated formation of ROS in non–bone marrow (BM)-derived liver cells plays a key role in liver fibrosis.
2′7′-Dichlorofluorescein diacetate (CM-H2 DCFDA) was purchased from Molecular Probes, Inc. (Eugene, OR). Palmitic acid was purchased from Sigma Aldrich (St. Louis, MO).
Animals and Treatment.
p47phox-Deficient mice on a C57BL/6 background, which lack a critical cytosolic component required for assembly of an active NOX complex, and p47phox-sufficient wild-type (WT) C57BL/6 control mice were purchased from Taconic Corp. (Hudson, NY).22 Six-week-old to eight-week-old male mice were used for liver injury experiments and for diet treatment. Liver fibrosis was induced either by bile duct ligation (BDL) or by intraperitoneal injection of the hepatotoxin carbon tetrachloride (CCl4). Mice were housed in a pathogen-free barrier facility accredited by the Association for the Accreditation and Assessment of Laboratory Animal Care.
Mice were fed ad libitum a high-fat, methionine-choline-deficient (MCD) diet (ICN Biomedicals, Sydney, Australia) for up to 10 weeks.23 Controls were pair-fed the same diet supplemented with choline chloride (2 g/kg) and D,L-methionine (3 g/kg) (MCS diet).
Mouse HEPs were isolated from WT and P47phox-deficient mice as described.9, 11 HEPs were subsequently plated on dishes coated with type I collagen and cultured in Waymouth's medium (GIBCO BRL; Life Technologies) containing 10% fetal bovine serum, 0.1 mmol/L insulin, and 0.1 mmol/L dexamethasone. After 2 hours, the cultures were washed with phosphate-buffered saline and changed to Roswell Park Memorial Institute medium (GIBCO BRL). HEPs were incubated with agonists for an additional 12 hours.24 HEPs, KCs, endothelial cells, fibrocytes, and HSCs were isolated from control and BDL mice, as described.9, 25, 26
Paraffin-embedded sections were stained with hematoxylin & eosin and Sirius red. For immunohistochemical analysis, sections were deparaffinized, rehydrated, and stained using the DAKO EnVision system protocol (DAKO, Carpinteria, CA). Sections were incubated with anti–alpha-smooth muscle actin (αSMA) (1:1000; DAKO) or 4-hydroxynonenal (4-HNE) for 30 minutes at room temperature. As negative controls, all specimens were incubated with an isotype-matched control antibody. The area of positive staining was measured using a computer-based morphometric analysis system. For immunofluorescence analysis, frozen sections were incubated with antibodies for 4-HNE, αSMA (DAKO), F4/80 (eBioscience), or pan-cytokeratin (Biolegend) overnight.
Quantification of Hepatic Collagen Content.
Collagen content was assessed by both morphometric analysis of Sirius red staining of liver sections and by hydroxyproline concentration. The area of positive Sirius red staining was measured using a computerized analysis method. Hydroxyproline content was quantified colorimetrically from 0.1 g liver samples.
Measurement of Intracellular ROS.
Cells cultured in 24-well plates were loaded with the redox-sensitive dye DCFDA (10 μM) for 20 minutes at 37°C. Cells were then stimulated with an agonist. DCFDA fluorescence was detected at excitation and emission wavelengths of 488 nm and 520 nm, respectively.9 ROS formation was measured using a multiwell fluorescence scanner (CytoFluor 2300; Millipore, Bedford, MA).
Liver extracts were obtained in a modified radioimmunoprecipitation buffer as described.18 Western blotting was performed using standard protocols. An antibody against αSMA (Sigma-Aldrich) was used at the concentration of 1:1000. Horseradish peroxidase–conjugated secondary antibodies were used and visualized with enhanced chemiluminescence.
Bone Marrow Transplantation.
Bone marrow transplantation (BMT) was performed as described.25 Mice received an intravenous injection of liposomal clodronate (200 μL intravenously) before irradiation to deplete KCs.27 Tibias and femurs of donor mice were flushed to obtain bone marrow (BM). BM cells (1 × 107) were injected into the tail veins of lethally irradiated (11 Gy) recipient mice. BDL was performed 12 weeks after BMT. To determine successful BMT in p47phox-deficient and p47phox-sufficient mice, spleen cells were isolated from BDL chimeric mice and analyzed by quantitative real-time polymerase chain reaction (RT-PCR) to measure p47phox messenger RNA (mRNA) expression (data not shown).
RT-PCR was used for measuring mRNA levels of fibrogenic markers (collagen α1(I) and αSMA). Extraction of RNA from total liver of mice was performed by a combination of TRIzol (Invitrogen, Carlsbad, CA) and RNeasy columns (Qiagen, Valencia, CA). Complementary DNA was obtained using the Amersham kit for complementary DNA synthesis.11
Results are expressed as mean ± standard error of the mean. The results were analyzed using the unpaired Student t test or the Newman-Keuls test. A P value < 0.05 was considered statistically significant.
p47phox-Deficient Mice Are Resistant to Liver Fibrosis Induced by BDL or CCl4.
To assess the role of the NOX in liver fibrosis, p47phox knockout (KO) mice were subjected to two different models of hepatic damage: BDL as a model of cholestatic liver injury and CCl4 treatment as a model of toxic liver injury. Consistent with our previous studies,9 mice deficient for the p47phox component of NOX had reduced fibrosis after 3 weeks of BDL, as evaluated by collagen deposition and αSMA staining (Fig. 1A,B). Furthermore, the critical role of NOX in liver fibrosis was confirmed in mice subjected to intraperitoneal injection of CCl4. Mice received 16 injections of CCl4 (0.5 μL/g body weight; twice weekly) and were sacrificed 2 days after the last injection. WT mice displayed a significant increase in collagen deposition and αSMA staining following treatment with CCl4 compared to vehicle-treated mice (Fig. 1C,D). The increase in fibrotic parameters was significantly reduced in p47phox-deficient mice (Fig. 1C,D). In addition, mRNA levels of collagen α1(I) and αSMA were significantly reduced in p47phox-deficient mice compared to p47phox-sufficient mice either after 3 weeks of BDL or after 16 injections of CCl4, as evaluated by RT-PCR (Fig. 1E,F). Moreover, the protein expression of αSMA was reduced in p47phox-deficient mice in comparison to p47phox-sufficient mice, either after BDL or CCl4 (Fig. 1E,F). These data demonstrate that NOX plays a crucial role in the pathogenesis of liver fibrosis in two complementary models of liver injury.
NOX in HSCs Plays a Crucial Role in Cholestatic Liver Injury.
To investigate the expression of NOX in different cell populations of the liver, we performed RT-PCR analysis for the mRNA levels of the main components of NOX activity in different cells of the liver: fibrocytes, KCs, HEPs, endothelial cells, and HSCs. The data showed that the p47phox component is mainly expressed in KCs, among the BM-derived cells, and in HSCs, among the non–BM-derived cells (Fig. 2A). Surprisingly, HEP expressed p47phox. Furthermore, the data showed a lower expression of this component in other cell types of the liver (Fig. 2A). Concordantly with the p47phox mRNA expression in the different hepatic cell types, we performed double immunofluorescence staining between 4-HNE (a lipid peroxidation product as a general marker of ROS) and cell-type markers F4/80, αSMA, and pan-cytokeratin for KCs, HSCs, and parenchymal cells, respectively. The lipid peroxidation product 4-HNE was expressed in KCs and HSCs in the livers of BDL mice (Fig. 2B,C). Some HEPs also express ROS in BDL mice (Fig. 2D). We further confirmed that these cell types express ROS in the livers of CCl4-treated mice (Supporting Fig. 1).
To investigate the role of NOX from different hepatic cell types in liver fibrosis, BMT was employed to generate chimeric mice. BM from p47phox-sufficient mice was transplanted into p47phox-deficient mice and vice versa, obtaining mice with NOX-sufficient BM-derived cells, including KCs and NOX-deficient endogenous liver cells (non–BM-derived), including HSCs, and vice versa. As controls, BM from p47phox-deficient mice was transplanted into p47phox-deficient mice and BM from p47phox-sufficient mice was transplanted into p47phox-sufficient mice. This strategy produced four different groups of mice: (1) WT BM → p47phox KO mice, (2) WT BM → WT mice, (3) p47phox KO BM → WT mice, and (4) p47phox KO BM → p47phox KO mice. We have previously shown that this strategy is useful in discriminating signaling pathways between HSCs and KCs.25 In summary, this resulted in mice with HSCs deficient for p47phox but with normal KCs (group 1), mice with intact NOX in all cells (group 2), mice with NOX-deficient KCs but WT HSCs (group 3), and mice deficient for NOX in all cells (group 4). These mice were then subjected to BDL for 3 weeks.
As expected from our above results, mice with intact NOX (group 2) showed the most fibrosis and mice deficient for p47phox in all cells (KO → KO, group 4) showed the least fibrosis (Fig. 3A-C). Deficiency for p47phox in either BM-derived or non–BM-derived cells resulted in attenuated liver fibrosis following 3 weeks of BDL as evaluated by collagen deposition and hydroxyproline content compared to mice with functional p47phox in all cells (WT BM → WT mice, group 2) (Fig. 3A-C). Furthermore, chimeric mice with NOX-deficient endogenous liver cells, including HSCs, but WT BM-derived cells, including KCs (WT BM → p47phox KO mice, group 1), showed reduced levels of collagen deposition and hydroxyproline content in comparison to chimeric mice with NOX-sufficient endogenous liver cells, including HSCs, but NOX-deficient BM-derived cells (p47phox KO BM → WT mice, group 3) (Fig. 3A-C). Accordingly, chimeric mice with NOX-deficient endogenous liver cells but WT BM-derived cells showed a significant reduction of αSMA expression, as demonstrated by immunohistochemistry and western blotting (Fig. 3D,E). Moreover, mRNA expression for αSMA and collagen α1(I) confirmed the reduced expression of fibrogenic markers in chimeric mice with NOX-deficient HSCs (Fig. 3F). These results suggest that NOX-mediated generation of ROS in endogenous liver cells, including HSCs, is more important than in BM-derived cells, including KCs, for the development of fibrosis following cholestatic liver injury.
Chimeric Mice with NOX-Deficient Endogenous Liver Cells Showed Reduced Peroxidation.
NOX generates ROS in many cell types. To investigate the levels of peroxidation in the NOX-chimeric livers, mice subjected to BMT were analyzed for peroxidation by immunohistochemistry for hydroxynonenal adducts. As expected, NOX-deficient mice showed reduced peroxidation in comparison to NOX-sufficient mice. Interestingly, chimeric mice with NOX-deficient HSCs showed a reduced level of peroxidation, confirming the importance of oxidative stress produced by NOX in HSCs during the process of liver fibrosis. (Fig. 4A).
Peroxidation was also measured in whole liver samples by thiobarbituric acid reactive substances (TBARS) assays. Peroxidation in chimeric livers with NOX-deficient HSCs had a greater reduction in lipid peroxidation than the chimeric livers with NOX-deficient KCs. In fact, the level of peroxidation produced by these chimeric mice was similar to the peroxidation in complete p47phox KO mice (Fig. 4B).
To better differentiate the ROS activity in the different cell types in the liver, we performed double immunofluorescence for 4-HNE and αSMA in the chimeric mice (Fig. 4C,D). The experiment showed a colocalization of ROS production (4-HNE stain) and HSCs in chimeric mice with p47phox KO BM (p47phox KO BM → WT mice) subjected to BDL (Fig. 4C), whereas HSCs express little ROS in the chimeric mice with p47phox KO endogenous liver cells (WT BM → p47phox KO mice) subjected to BDL (Fig. 4D), suggesting that NOX is a major contributor in HSCs.
MCD Diet Induces Hepatic Steatosis Independent of NADPH Oxidase.
To investigate the role of NOX in a mouse model of nonalcoholic steatohepatitis (NASH) ultimately leading to fibrosis, NOX-deficient (p47phox KO) mice and WT controls were fed an MCD diet for 10 weeks. Although both WT and KO mice fed the MCD diet lost weight, the liver weight–body weight fraction revealed an increase in steatosis of the liver of all MCD-treated mice (Fig. 5A). In addition, the serum aminotransferase levels were significantly higher both in WT and KO mice fed the MCD diet than the MCS diet (Fig. 5B), suggesting an equal degree of steatosis and of hepatocellular damage both in WT and in KO mice. To confirm these results, RT-PCR for the mRNA of inflammatory markers such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNFα) showed no significant differences in WT and KO mice fed the MCD diet (Fig. 5C). However, there was significant difference in the mRNA expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), a marker of HSC activation (Fig. 5C).
Livers from mice fed an MCD diet showed significant deposition of triglycerides, with macronodular and micronodular distribution of fat as evaluated by Oil-red O staining compared to mice fed a control diet supplemented with methionine and choline (MCS diet; Fig. 5D,E). The higher deposition of fat in mice fed the MCD diet was confirmed by measuring hepatic triglycerides content (Fig. 5F). A slight increase in fat deposition in mice receiving the MCS control diet was observed compared to mice fed a normal chow diet (Fig. 5D,E). There was no difference in hepatic lipid content between NOX-deficient and WT mice (Fig. 5D-F). This suggests that NOX is dispensable for fat accumulation in a mouse model of nonalcoholic fatty liver disease (NAFLD).
MCD Diet Induces Similar Levels of Peroxidation in the Livers of WT and NOX-Deficient Mice.
Oxidative stress is a hallmark of NASH, which is recapitulated in mice fed an MCD diet. Reductions in antioxidant defense mechanisms as well as increases in ROS production are attributed to methionine-choline deficiency. Immunohistochemistry for 4-HNE adducts showed similar staining in the livers of NOX-deficient (p47phox KO) and WT mice fed an MCD diet, indicating a diet-induced increase in ROS production that is independent of NOX (Fig. 6A). Accordingly, TBARS levels were significantly increased in mice fed an MCD diet compared to an MCS diet, but no difference between WT and KO mice was observed (Fig. 6B). This result was confirmed by immunofluorescence staining in MCS-fed and MCD-fed mice that revealed 4-HNE in the hepatocytes of both WT and KO MCD-fed mice (Fig. 6C). Thus, the generation of total hepatic ROS in this NASH model is independent of NOX.
NOX Is Critical for the Development of MCD Diet-Induced Liver Fibrosis.
Even if NOX does not affect fat accumulation and generation of total hepatic ROS, it might still affect the development of liver fibrosis following an MCD diet. Sirius red staining indicated that feeding an MCD diet for 10 weeks results in significant fibrosis in WT mice compared to mice fed an MCS diet. However, NOX-deficient (p47phox KO) mice fed an MCD diet failed to develop fibrosis (Fig. 7A). Collagen α1(I) and αSMA mRNAs were increased in WT mice fed an MCD diet in comparison to mice fed the control MCS diet. This increase in fibrogenic markers was significantly attenuated in NOX-deficient mice fed an MCD diet (Fig. 7B). Collectively, these data suggest that NOX is not involved in lipid metabolism and hepatic fat accumulation but NOX is required for the development of fibrosis in the metabolic model of liver disease. Indeed, HSCs express ROS in WT, but not NOX-deficient, mice fed an MCD diet (Supporting Fig. 2)
Free Fatty Acid–Induced ROS Production in HEPs Is NOX-Independent.
Our data suggest that the MCD diet causes steatosis and oxidative stress independent of NOX, but that the subsequent development of fibrosis in this model of metabolic liver injury relies on NOX. To further evaluate the role of NOX in HEPs, we isolated primary HEPs from NOX-deficient (p47phox KO) and WT mice and incubated them with palmitic acid (200 μM) for 12 hours. Oil-red O staining showed a similar extent of triglyceride accumulation in WT and NOX-deficient HEPs (Fig. 8A). Moreover, incubation with palmitic acid caused a significant increase in ROS production as evaluated by DCFDA measurement in comparison to untreated cells (Fig. 8B). However, increases in ROS production were similar between WT and p47phox-deficient HEPs (Fig. 8B). Thus, NOX is not required for free fatty acid (FFA)-induced triglyceride accumulation and ROS generation in HEPs.
NOX is a multiprotein complex that generates ROS in response to a wide range of stimuli.28 In the liver, NOX is expressed in both phagocytic and nonphagocytic forms. Chronic liver diseases are characterized by increased ROS production as well as decreased activity of antioxidant systems, resulting in oxidative stress.29 This feature is commonly detected in patients with alcoholic liver disease, hepatitis C virus infection, hemochromatosis, and cholestatic liver diseases. Similar observations were made in experimental models of liver fibrosis. Oxidative stress is not only a consequence of chronic liver injury but also significantly contributes to excessive tissue remodeling and fibrogenesis.30 NOX has emerged as a primary source of ROS in liver disease. KCs in the liver mainly produce ROS through the phagocytic form of NOX,16 which exerts an important role in host defense and inflammation.31 HSCs express a nonphagocytic form of NOX, which plays a critical role in activating signaling pathways.9, 32 Fibrogenic agonists such as angiotensin II, leptin, platelet-derived growth factor, and apoptotic bodies activate NOX in cultured HSCs.9, 17, 18, 20, 33 Both pharmacological inhibition with diphenyleneiodonium34 and genetic studies using p47phox-deficient mice provided evidence for a central role of NOX in the regulation of HSC activation and liver fibrosis. Whereas there are convincing data regarding the in vitro activation of HSCs,35 the contribution of NOX in different hepatic cell types to hepatic fibrogenesis in vivo remains mostly unknown. Because KCs are the major source of phagocytic NOX in the liver and are required for HSC activation and fibrogenesis,36 it could be possible that phagocytic NOX is the main mediator of hepatic fibrogenesis,37 or that both KCs and HSCs mediate fibrogenesis.
Our study used BMT to compare the contribution of NOX-derived ROS from BM-derived cells, including KCs, and endogenous liver cells, including HSCs, to hepatic fibrosis. Our data show that NOX in both BM-derived cells and endogenous liver cells contributes to liver fibrogenesis. Earlier studies proposed that the NOX in KCs was critical for alcoholic liver disease.38 However, this model produced steatosis and inflammation, but not fibrosis. In fact, the current study demonstrated that chimeric mice with NOX-deficient HSCs but WT KCs had the greatest reduction in liver fibrosis. The possibility of creating a selective inhibition of the nonphagocytic form of NOX39-41 without the involvement of the phagocytic form should significantly reduce the fibrogenic pathway without affecting host defense mechanisms related to the functionality of the phagocytic form of NOX.42
NAFLD, the liver manifestation of the metabolic syndrome, may progress to liver fibrosis and cirrhosis.43 Moreover, although the main source of ROS production in both viral and ethanol-induced liver injury appears to result from activation of NOX,44-46 the role of NOX in NAFLD is still unclear. In fact, the main cell types involved in ROS production during NAFLD are perhaps HEPs.47 HEPs express a functional form of NOX that participates in CD95-induced cell death.21 Our study demonstrates that the development of steatosis, lipid peroxidation, and inflammation caused by an MCD diet are independent from the p47 subunit of the NOX. This conclusion was supported by data showing the same triglyceride accumulation and ROS in primary cultures of HEPs isolated from p47phox KO and WT mice. In fact, the majority of ROS production in MCD-induced liver injury is derived from hepatocellular lipid deposition and subsequent peroxidation. Other sources of ROS in HEPs are the cytochrome P450s and mitochondrial respiratory chain.48, 49
However, our study revealed that NOX does play a role in the steatosis–inflammation–fibrosis axis in NAFLD, in that NOX-deficient mice express little ROS in HSCs, and develop less fibrosis compared to WT mice on an MCD diet for 10 weeks (Fig. 7; Supporting Fig. 2). Thus, NOX was required for ROS generation in HSCs and fibrosis but not steatosis or ROS generation in HEPs in this model of NAFLD. However, because the MCD diet is not as robust in inducing liver fibrosis as BDL or CCl4, we could not perform the same chimeric liver studies to further identify the key cell types expressing NOX in the NASH model.
Another mechanism of fibrogenesis is represented by apoptosis and then phagocytosis of apoptotic bodies.33 Apoptotic bodies may directly or indirectly, through KCs, activate HSCs and promote myofibroblastic transdifferentiation. NOX plays a critical role in the process of phagocytosis in response to apoptotic bodies that are generated during liver injury. Thus, the reduced fibrosis observed in p47phox KO mice may be related to the inhibition of the fibrogenic mechanism induced by apoptotic bodies.
In conclusion, our study points to a crucial role of nonphagocytic NOX in liver fibrosis but not steatosis in experimental liver fibrosis including NAFLD. Thus, not all ROS is the same, so that ROS generated by NOX in HSCs is fibrogenic, whereas ROS generated in steatotic hepatocytes is NOX-independent. Specific inhibition of nonphagocytic NOX components may inhibit fibrosis without interfering with the immune host defenses, and thus represents a promising pharmacological target.