Volume 139, Issue 4, Pages 1375-1384.e4 (October 2010) Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 Plays a Key Role in Stellate Cell Activation and Liver Fibrogenesis In Vivo  Joy X. Jiang, Senthil Venugopal, Nobuko Serizawa, Xiangling Chen, Fiona Scott, Yong Li, Roger Adamson, Sridevi Devaraj, Vijay Shah, M. Eric Gershwin, Scott L. Friedman, Natalie J. Török  Gastroenterology  Volume 139, Issue 4, Pages 1375-1384.e4 (October 2010) DOI: 10.1053/j.gastro.2010.05.074 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 The expression of NOX2 is up-regulated in HSC during liver fibrogenesis. (A) Myofibroblasts in patients with hepatitis C virus (HCV) (a, b, c) and primary biliary cirrhosis (PBC) (d, e, f) express NOX2 (green channel, a, d), α-SMA (red channel, b, e), and colocalization images (c, f) by confocal microscopy. Scale bar, 30 μm. (B) HSC were isolated from BDL and sham rats, and NOX2 messenger RNA (mRNA) and protein expression were assessed. Both NOX2 mRNA and protein (*P < .05) were significantly up-regulated compared with HSC from sham-operated animals (shown in fold expression). (C) HSC were isolated from rats and exposed to AB for 24 hours. NOX2 expression was determined by real-time PCR. Phagocytosis of AB induced NOX2 up-regulation in HSC, shown as fold increase (**P ≤ .01). Error bars indicate 1 SED. Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 NOX2 activation in HSC results in superoxide production and up-regulation of collagen I messenger RNA by inducing its transcriptional activation. (A) Primary rat HSC were treated with either scrambled siRNA or NOX2 siRNA and exposed to AB. After 6 hours, superoxide production was assessed by the lucigenin assay. In the scrambled siRNA-transfected HSC, phagocytosis induced superoxide production, and this was inhibited in the NOX2 siRNA-transfected cells (NT, nontransfected; C, control, not exposed to AB; *P < .05, average of 4 experiments; bars indicate 1 SED, shown in fold expression). (B) Collagen IA1 expression was assessed by real-time PCR. Collagen IA1 expression was up-regulated in the scrambled siRNA-transfected cells after AB exposure (shown in fold expression, **P ≤ .01), and it decreased significantly in the NOX2 siRNA-transfected primary HSC (##P ≤ .0001). (C) Primary wild-type or NOX2−/− HSC were transfected by constructs containing the truncated collagen promoter Col1A2 P1-Luc (−378/+58) or with a construct where the peroxide-responsive area is intact (Col1A2 P1-Luc [−2900/+58]) or the empty vector. The cells were exposed to AB in the presence or absence of the reducing agent GSH or catalase. Phagocytosis resulted in a significant induction of the COLIA2 promoter activity in the Col1A2 P1-Luc (−2900/+58)-transfected cells. This was abrogated in the Col1A2 P1-Luc (−378/+58)-transfected cells or decreased after exposure to catalase or GSH. In wt cells, the collagen promoter activity thus resulted from NOX2-mediated peroxide production following phagocytosis. In the NOX2−/− cells, the luciferase activity was significantly blunted after exposure to AB (C, control cells; AB, apoptotic bodies; cata, catalase; GSH, glutathione. *P < .05, **P ≤ .01. Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 The phagocytic activity is decreased in NOX2−/− HSC. (A) Primary wt or NOX2−/− HSC were exposed to carboxytetramethyl rhodamine succinimidyl ester-labeled AB, and the rate of phagocytosis was assessed after 24 hours. The engulfment was significantly decreased in HSC isolated from NOX2−/− mice compared with wt mice (#P < .001). (B) Pull down of active GTP-bound Rac1 from the membrane fractions of wt and NOX2−/− HSC demonstrated significantly more GTP-Rac1 in the membrane fraction of wt cells following AB exposure compared with NOX2−/− cells. Rac1 immunostaining was detectable at the phagosomal membrane forming around the carboxytetramethyl rhodamine succinimidyl ester-labeled AB (red) in wt HSC (arrow), whereas in the NOX2−/− HSC, AB attachment is seen but impaired phagosome formation (arrowhead). Scale bar, 10 μm. Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 In vivo model of hepatocyte apoptosis and phagocytosis. To follow the fate of apoptotic hepatocytes, mice were first injected with the α1-AT-LV-GFP via the portal vein, and, then 7 days later, Ad-TRAIL was injected into the tail vein of the same mice. As control, mice were injected only with Ad-TRAIL or Ad-GFP (images not shown) or with α1-AT-LV-GFP. To inhibit apoptosis, a separate group of mice were injected with Q-VD-OPH, pancaspase inhibitor before and after the Ad-TRAIL injection. The liver from only Ad-TRAIL or LV-injected mice showed no significant injury or infiltration by inflammatory cells, and the ALT values remained normal (Figure 4b, c). In the LV plus Ad-TRAIL-injected animals, the liver showed mild to moderate hepatocyte injury, increased ALT values (*P < .05), and no significant infiltration by inflammatory cells (Figure 4d). In the Q-VD-OPH treated animals, the liver showed normal histology (Figure 4e) and decreased ALT values (*P < .05) compared with LV plus Ad-TRAIL-injected mice. To assess apoptosis, TUNEL assays were done on all liver samples. In the LV plus Ad-TRAIL infected livers, hepatocyte apoptosis was increased (Figure 4d') compared with only Ad-TRAIL (Figure 4b') or only LV (Figure 4c')-injected animals. In the Q-VD-OPH-treated animals, apoptosis decreased (Figure 4e'). Scale bar, 50 μm. Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Phagocytosis of apoptotic hepatocytes in vivo results in a fibrogenic response. Immunohistochemistry and confocal microscopy were performed to visualize GFP-labeled hepatocytes and their AB and activated HSC (α-SMA, red). (GFP was only expressed in hepatocytes because of the hepatocyte specific α1-AT promoter). There was an increase in the number of activated HSC (α-SMA, red) in the liver of mice injected with α1-AT-LV-GFP plus Ad-TRAIL (Figure 5D, E), whereas the only Ad-TRAIL (Figure 5B) or only LV-GFP (Figure 5C) injected mice had the same amount of activated HSC as control, uninjected mice (Figure 5A). Scale bar, 50 μm. At higher magnification in the α-SMA-positive HSC, GFP-labeled AB could be seen, indicating phagocytosis of hepatocyte-derived AB (arrow, arrowhead depict apoptotic hepatocytes and AB, respectively, Figure 5F). Scale bar, 20 μm. Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 NOX2−/− mice exhibit decreased profibrogenic activity (A). Wt and NOX2−/− mice were injected with Ad-TRAIL or LV or LV plus Ad-TRAIL. Immunohistochemistry showed much less α-SMA positive HSC in NOX2−/− mice after injection of LV plus Ad-TRAIL (Figure 6A, a), suggesting that in these animals less HSC activation had occurred (scale bar, 50 μm). In wt animals treated with Q-VD-OPH, fewer α-SMA positive HSC are seen (Figure 6A, b). Real-time PCR was performed from the liver tissues of wt and NOX2−/− mice in the above experiments using α-SMA, collagen IA1, and TGF-β1-specific primers (B). The expression of the fibrosis-related transcripts had increased in wt Ad-TRAIL plus LV-injected mice, whereas no increase was detected in NOX2−/− animals (*P < .05, **P < .01). In wt animals treated with Q-VD-OPH, the increase in α-SMA expression was significantly lower than in vehicle treated wt mice (*P ≤ .05), and collagen IA1 and TGF-β1 levels decreased significantly, as well (*P ≤ .05). Data expressed as fold over control (control mice, not injected). Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Liver fibrosis is decreased in NOX2−/− mice. (A) Wt or NOX2−/− mice were bile duct ligated (BDL) then killed after 3 weeks (in the case of some NOX2−/− mice after 6 weeks) following surgery. Picrosirius staining was done to assess fibrosis stage. In NOX2−/− animals, the fibrosis stage was significantly lower compared with that of wt animals following BDL (Figure 7A, b, c, d). NOX2−/− mice survived longer with less fibrosis compared with wt animals (Figure 7A, b, d). ALT and bilirubin values were increased in both the wt and NOX2−/− BDL animals (*P < .05). (B) Mice having undergone BDL were injected with GdCl3, throughout the experiment, to inhibit macrophage function. The fibrosis decreased in GdCl3-injected wt animals after BDL (Figure 7B, a) compared with phosphate-buffered saline (PBS)-injected controls (Figure 7A, b), whereas no significant change was seen in NOX2−/− livers; scale bars, 50 μm. Real-time PCR was performed using collagen IA1 specific primers and OH-proline incorporation assay to assess the amount of collagen in all samples. In NOX2−/− BDL mice, the expression of collagen IA1 (**P ≤ .01) and OH-proline incorporation significantly decreased (*P < .05) compared with wt BDL animals. Inhibiting macrophages decreased collagen IA1 expression (*P < .05) and OH-proline incorporation (*P < .05) in wt BDL animals, compared with PBS-injected animals, but not to the extent seen in NOX2−/− BDL mice. In GdCl3-injected mice, the ALT values were overall lower (*P < .05) than in PBS-injected mice; however, the values among GdCl3-injected wt and NOX2−/− animals were not significantly different. Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Inflammatory markers Cd11b and tumor necrosis factor α (TNF-α) expression are not increased following lentiviral and Ad-TRAIL injection. To assess for possible recruitment of inflammatory cells, immunohistochemistry was done using an anti-CD11b antibody. There were only rare positive cells in all experimental conditions, and the total number of cells counted in each condition was not significantly different (data not shown). Insert shows a Cd11b positive cell at a higher magnification (arrowhead). Real-time PCR was done to assess TNF-α expression, and there was no statistical difference found (in LV-injected wt mice, the TNF-α expression was lower, but this was not statistically significant). Gastroenterology 2010 139, 1375-1384.e4DOI: (10.1053/j.gastro.2010.05.074) Copyright © 2010 AGA Institute Terms and Conditions