LIGHT is an important costimulatory molecule for T cell immunity. Recent studies have further implicated its role in innate immunity and inflammatory diseases, but its cellular and molecular mechanisms remain elusive. We report here that LIGHT is upregulated and functions as a proinflammatory cytokine in 2 independent experimental hepatitis models, induced by concanavalin A and Listeria monocytogenes. Molecular mutagenesis studies suggest that soluble LIGHT protein produced by cleavage from the cell membrane plays an important role in this effect through the interaction with the lymphotoxin-β receptor (LTβR) but not herpes virus entry mediator. NK1.1+ T cells contribute to the production, but not the cleavage or effector functions, of soluble LIGHT. Importantly, treatment with a mAb that specifically interferes with the LIGHT-LTβR interaction protects mice from lethal hepatitis. Our studies thus identify a what we believe to be a novel function of soluble LIGHT in vivo and offer a potential target for therapeutic interventions in hepatic inflammatory diseases.
Sudarshan Anand, Pu Wang, Kiyoshi Yoshimura, In-Hak Choi, Anja Hilliard, Youhai H. Chen, Chyung-Ru Wang, Richard Schulick, Andrew S. Flies, Dallas B. Flies, Gefeng Zhu, Yanhui Xu, Drew M. Pardoll, Lieping Chen, Koji Tamada
Aniela Jakubowski, Christine Ambrose, Michael Parr, John M. Lincecum, Monica Z. Wang, Timothy S. Zheng, Beth Browning, Jennifer S. Michaelson, Manfred Baetscher, Bruce Wang, D. Montgomery Bissell, Linda C. Burkly
Progenitor (“oval”) cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.
Aniela Jakubowski, Christine Ambrose, Michael Parr, John M. Lincecum, Monica Z. Wang, Timothy S. Zheng, Beth Browning, Jennifer S. Michaelson, Manfred Baestcher, Bruce Wang, D. Montgomery Bissell, Linda C. Burkly
Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of excess liver triacylglycerol (TAG), inflammation, and liver damage. The goal of the present study was to directly quantify the biological sources of hepatic and plasma lipoprotein TAG in NAFLD. Patients (5 male and 4 female; 44 ± 10 years of age) scheduled for a medically indicated liver biopsy were infused with and orally fed stable isotopes for 4 days to label and track serum nonesterified fatty acids (NEFAs), dietary fatty acids, and those derived from the de novo lipogenesis (DNL) pathway, present in liver tissue and lipoprotein TAG. Hepatic and lipoprotein TAG fatty acids were analyzed by gas chromatography/mass spectrometry. NAFLD patients were obese, with fasting hypertriglyceridemia and hyperinsulinemia. Of the TAG accounted for in liver, 59.0% ± 9.9% of TAG arose from NEFAs; 26.1% ± 6.7%, from DNL; and 14.9% ± 7.0%, from the diet. The pattern of labeling in VLDL was similar to that in liver, and throughout the 4 days of labeling, the liver demonstrated reciprocal use of adipose and dietary fatty acids. DNL was elevated in the fasting state and demonstrated no diurnal variation. These quantitative metabolic data document that both elevated peripheral fatty acids and DNL contribute to the accumulation of hepatic and lipoprotein fat in NAFLD.
Kerry L. Donnelly, Coleman I. Smith, Sarah J. Schwarzenberg, Jose Jessurun, Mark D. Boldt, Elizabeth J. Parks
The inhibitor of NF-κB (I-κB) kinase (IKK) complex consists of 3 subunits, IKK1, IKK2, and NF-κB essential modulator (NEMO), and is involved in the activation of NF-κB by various stimuli. IKK2 or NEMO constitutive knockout mice die during embryogenesis as a result of massive hepatic apoptosis. Therefore, we examined the role of IKK2 in TNF-induced apoptosis and ischemia/reperfusion (I/R) injury in the liver by using conditional knockout mice. Hepatocyte-specific ablation of IKK2 did not lead to impaired activation of NF-κB or increased apoptosis after TNF-α stimulation whereas conditional NEMO knockout resulted in complete block of NF-κB activation and massive hepatocyte apoptosis. In a model of partial hepatic I/R injury, mice lacking IKK2 in hepatocytes displayed significantly reduced liver necrosis and inflammation than wild-type mice. AS602868, a novel chemical inhibitor of IKK2, protected mice from liver injury due to I/R without sensitizing them toward TNF-induced apoptosis and could therefore emerge as a new pharmacological therapy for liver resection, hemorrhagic shock, or transplantation surgery.
Tom Luedde, Ulrike Assmus, Torsten Wüstefeld, Andreas Meyer zu Vilsendorf, Tania Roskams, Mark Schmidt-Supprian, Klaus Rajewsky, David A. Brenner, Michael P. Manns, Manolis Pasparakis, Christian Trautwein
Increasing evidence demonstrates that IL-6 has a protective role during liver injury. IL-6 activates intracellular pathways via the gp130 receptor. In order to identify IL-6–gp130 pathways involved in mediating liver protection, we analyzed hepatocyte-specific gp130 knockout mice in a concanavalin A–induced (Con A–induced) model of immune-mediated hepatitis. We demonstrated that IL-6–gp130–dependent pathways in hepatocytes alone are sufficient for triggering protection in Con A–induced hepatitis. gp130-STAT3 signaling in hepatocytes mediates the IL-6–triggered protective effect. This was demonstrated by analysis of IL-6–induced protection in mice selectively deficient for gp130-dependent STAT1/3 or gp130-SHP2-RAS signaling in hepatocytes. To identify IL-6–gp130–STAT1/3 dependently expressed liver-protective factors, we performed gene array analysis of hepatic gene expression in hepatocyte-specific gp130–/– mice as well as in gp130-STAT1/3– and gp130-SHP2-RAS-MAPK–deficient mice. The mouse IL-8 ortholog KC (also known as Gro-α) and serum amyloid A2 (SAA2) was identified as differentially IL-6–gp130–STAT3–regulated genes. Hepatic expression of KC and SAA2 mediate the liver-protective potential of IL-6, since treatment with recombinant KC or serum SAA2 effectively reduced liver injury during Con A–induced hepatitis. In summary, this study defines IL-6–gp130–STAT3–dependent gene expression in hepatocytes that mediates IL-6–triggered protection in immune-mediated Con A–induced hepatitis. Additionally, we identified the IL-6–gp130–STAT3–dependent proteins KC and SAA2 as new candidates for therapeutic targets in liver diseases.
Christian Klein, Torsten Wüstefeld, Ulrike Assmus, Tania Roskams, Stefan Rose-John, Michael Müller, Michael P. Manns, Mattias Ernst, Christian Trautwein
Cholecystokinin (CCK) modulates contractility of the gallbladder, the sphincter of Oddi, and the stomach. These effects are mediated through activation of gastrointestinal smooth muscle as well as enteric neuron CCK-1 receptors (CCK-1Rs). To investigate the potential physiological and pathophysiological functions linked to CCK-1R–mediated signaling, we compared male WT and CCK-1R–deficient mice (129/SvEv). After 12 weeks on either a standard mouse chow or a lithogenic diet (containing 1% cholesterol, 0.5% cholic acid, and 15% dairy fat), small-intestinal transit time, intestinal cholesterol absorption, biliary cholesterol secretion, and cholesterol gallstone prevalence were compared in knockout versus WT animals. Analysis of mice on either the chow or the lithogenic diet revealed that CCK-1R–/– animals had larger gallbladder volumes (predisposing to bile stasis), significant retardation of small-intestinal transit times (resulting in increased cholesterol absorption), and increased biliary cholesterol secretion rates. The elevation in bile cholesterol, coupled with a tendency toward gallbladder stasis (due to the absence of CCK-induced contraction), facilitates nucleation, growth, and agglomeration of cholesterol monohydrate crystals; this sequence of events in turn results in a significantly higher prevalence of cholesterol gallstones in the CCK-1R–null mice.
David Q.-H. Wang, Frank Schmitz, Alan S. Kopin, Martin C. Carey
The etiology and pathogenesis of bile duct obstruction in children with biliary atresia are largely unknown. We have previously reported that, despite phenotypic heterogeneity, genomic signatures of livers from patients display a proinflammatory phenotype. Here, we address the hypothesis that production of IFN-γ is a key pathogenic mechanism of disease using a mouse model of rotavirus-induced biliary atresia. We found that rotavirus infection of neonatal mice has a unique tropism to bile duct cells, and it triggers a hepatobiliary inflammation by IFN-γ–producing CD4+ and CD8+ lymphocytes. The inflammation is tissue specific, resulting in progressive jaundice, growth failure, and greater than 90% mortality due to obstruction of extrahepatic bile ducts. In this model, the genetic loss of IFN-γ did not alter the onset of jaundice, but it remarkably suppressed the tissue-specific targeting of T lymphocytes and completely prevented the inflammatory and fibrosing obstruction of extrahepatic bile ducts. As a consequence, jaundice resolved, and long-term survival improved to greater than 80%. Notably, administration of recombinant IFN-γ led to recurrence of bile duct obstruction following rotavirus infection of IFN-γ–deficient mice. Thus, IFN-γ–driven obstruction of bile ducts is a key pathogenic mechanism of disease and may constitute a therapeutic target to block disease progression in patients with biliary atresia.
Pranavkumar Shivakumar, Kathleen M. Campbell, Gregg E. Sabla, Alexander Miethke, Greg Tiao, Monica M. McNeal, Richard L. Ward, Jorge A. Bezerra
Ali Canbay, Maria Eugenia Guicciardi, Hajime Higuchi, Ariel Feldstein, Steven F. Bronk, Robert Rydzewski, Makiko Tanai, Gregory J. Gores
We recently showed that antigen-nonspecific inflammatory cells are recruited into the liver when hepatitis B virus (HBV)-specific CTLs are injected into HBV transgenic mice, and that this process amplifies the severity of liver disease. We also showed that the severity of CTL-induced liver disease is ameliorated by the depletion of Gr-1+ cells (Gr-1 is an antigen highly expressed by neutrophils), which, secondarily, abolishes the intrahepatic recruitment of all antigen-nonspecific Gr-1– mononuclear cells (NK and NKT cells, T and B lymphocytes, monocytes, macrophages, dendritic cells) despite the strong induction of chemokine gene expression. Those results suggested that in addition to chemokine expression, CTL-induced functions are necessary for mononuclear cell recruitment to occur. We now report that MMPs known to be produced by Gr-1+ cells are rapidly induced in the livers of CTL-injected mice. The inhibition of MMP activity reduced the intrahepatic recruitment of antigen-nonspecific mononuclear cells and much of the attending liver disease without affecting the migration or antiviral potential of antigen-specific CTLs. The notion that the inhibition of MMP activity is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.
Giovanni Sitia, Masanori Isogawa, Matteo Iannacone, Iain L. Campbell, Francis V. Chisari, Luca G. Guidotti