Kupffer cell and interleukin-12-dependent loss of natural killer T cells in hepatosteatosis.

UNLABELLED: Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-12, major T helper (Th) 1 cytokines, and reduced hepatic natural killer T (NKT) cell numbers. The relationship between lipid accumulation, cytokine expression, and hepatic NKT cells is not known. This study was conducted to assess the role of IL-12 in the development of hepatic steatosis and its potential impact on liver NKT cells. Male C57Bl/6 wildtype (WT) and IL-12-deficient (IL-12(-/-)) mice were fed a choline-deficient diet (CDD) for 0, 10, or 20 weeks. CDD led to marked hepatosteatosis, reduced hepatic but not splenic NKT cell numbers and function, and increased hepatic expression of the T(h)1-type cytokines IL-12, interferon gamma (IFN-gamma), and TNF-alpha in WT mice. The absence of IL-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN-gamma and TNF-alpha expression. Treatment of CDD-fed mice with lipopolysaccharide led to a significant increase in hepatic IL-12 expression, and Kupffer cell (KC) depletion reduced liver IL-12 expression and restored NKT cells in CDD-induced fatty liver. Interestingly, KCs from CDD-fed mice failed to produce increased quantities of IL-12 upon activation in vitro when compared to similarly treated KCs from control fed mice, suggesting that secondary factors in vivo promote heightened IL-12 production. Finally, human livers with severe steatosis showed a substantial decrease in NKT cells. CONCLUSION: Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC-and IL-12-dependent manner. Our results suggest a pivotal and multifunctional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process that is likely active within human nonalcoholic fatty liver disease.

Accumulation of hedgehog-responsive progenitors parallels alcoholic liver disease severity in mice and humans.

BACKGROUND & AIMS: Improving outcomes in alcoholic liver disease (ALD) necessitates better understanding of how habitual ethanol (EtOH) consumption alters normal regenerative mechanisms within the liver. Hedgehog (Hh) pathway activation promotes expansion of progenitor populations in other tissues. We evaluated the hypothesis that chronic EtOH exposure activates Hh signaling in liver. METHODS: Hh signaling, liver progenitors, transforming growth factor (TGF)-beta induction, and liver damage were compared in mice fed chow, high-fat diets (HF), or HF + EtOH for 4 weeks. Susceptibility to TGF-beta-mediated apoptosis was compared in Hh-responsive liver cells (eg, immature cholangiocytes and oval cells) and mature hepatocytes (which are unresponsive to Hh). Hepatic accumulation of Hh-responsive cells were compared in controls and ALD patients and correlated with a discriminant function (DF) that predicts subacute mortality. RESULTS: Hh signaling and numbers of Hh-responsive cells were increased in HF mice and greatest in HF+EtOH mice. In both, progenitor and stromal cell populations harbored Hh-responsive cells. More ductular-type progenitors and fibrosis markers were noted in HF+EtOH mice than in HF mice. The former also expressed more TGF-beta-1. TGF-beta-1 treatment selectively promoted the viability of Hh-responsive immature liver cells and caused mature hepatocytes that survived to produce Hh ligands. Hh-responsive cells were increased in ALD patients. Lobular accumulation of Hh-responsive immature ductular cells was greater in those with a DF >32 than those with a DF <32. CONCLUSIONS: Hh signaling is increased in ALD and may influence ALD outcomes by promoting hepatic accumulation of immature ductular cells.

Pivotal role of Smad3 in a mouse model of T cell-mediated hepatitis.

UNLABELLED: Transforming growth factor beta (TGFbeta) promotes hepatocellular apoptosis and suppresses hepatic lymphocyte responses in part through activation of Smad3. The purpose of the current study was to determine the importance of Smad3 signaling in an experimental model of autoimmune hepatitis induced by concanavalin A (ConA), a process involving T cell activation and hepatocellular apoptosis. C57Bl/6 wild-type (Wt) or Smad3-deficient (Smad3(-/-)) mice were injected intravenously with 15 mg/kg ConA or vehicle. Nine hours post ConA injection, Wt mice presented with severe hepatitis as assessed by increased liver transferases. This injury was associated with eosinophil accumulation and preceded at 3 hours post-injection by significant increases in hepatic T helper 1 (interferon gamma) and T helper 2 (interleukin-4) cytokine production. Absence of Smad3 significantly blunted hepatocellular injury 9 hours post ConA injection, which was associated with reduced early T helper 1 and T helper 2 cytokine production and eosinophil accumulation. Smad3(-/-) livers also showed significant reductions in hepatocellular apoptosis as assessed by terminal UTP nick-end labeling when compared to ConA-treated Wt mice in conjunction with reduced caspase 3 cleavage, which was likely mediated by a Smad3-dependent inhibition of the survival factor extracellular signal-regulated kinase 1/2. In vitro, Smad3(-/-) hepatocytes were resistant to TGFbeta-induced apoptosis, and this protection was dependent on extracellular signal-regulated kinase activation. CONCLUSION: Together, these results show, for the first time, the significance of Smad3 signaling in autoimmune hepatitis, underlining the control of Smad3-dependent TGFbeta signaling on proinflammatory cytokine production, eosinophil recruitment, and hepatocellular apoptosis. Interruption of this pathway could be beneficial clinically to limit acute fulminant liver pathologies.

Impaired liver regeneration and increased oval cell numbers following T cell-mediated hepatitis.

UNLABELLED: The regeneration of liver tissue following transplantation is often complicated by inflammation and tissue damage induced by a number of factors, including ischemia and reperfusion injury and immune reactions to the donor tissue. The purpose of the current study is to characterize the effects of T cell-mediated hepatitis induced by concanavalin A (ConA) on the regenerative response in vivo. Liver regeneration following a partial (70%) hepatectomy (pHx) was associated with elevations in serum enzymes and the induction of key cell cycle proteins (cyclin D, cyclin E, and Stat3) and hepatocyte proliferation. The induction of T cell-mediated hepatitis 4 days before pHx increased serum enzymes 48 hours after pHx, reduced early cyclin D expression and Stat3 activation, and suppressed hepatocyte proliferation. This inhibition of proliferation was also associated with increased expression of p21, the activation of Smad2, the induction of transforming growth factor beta and interferon gamma expression, and reduced hepatic interleukin 6 production. Moreover, the ConA pretreatment increased the numbers of separate oval cell-like CD117(+) cells and hematopoietic-like Sca-1(+) cell populations 48 hours following pHx. The depletion of natural killer (NK) cells, an important component of the innate immune response, did not affect liver injury or ConA-induced impairment of hepatocyte proliferation but did increase the numbers of both CD117-positive and Sca-1-positive cell populations. Finally, splenocytes isolated from ConA-pretreated mice exerted cytotoxicity toward autologous bone marrow cells in an NK cell-dependent manner. CONCLUSION: T cell-mediated hepatitis alters early cytokine responses, reduces hepatocellular regeneration, and induces NK cell-sensitive oval cell and hematopoietic-like cell expansion following pHx.

LPS signaling enhances hepatic fibrogenesis caused by experimental cholestasis in mice.

Although it is clear that bile acid accumulation is the major initiator of fibrosis caused by cholestatic liver disease, endotoxemia is a common side effect. However, the depletion of hepatic macrophages with gadolinium chloride blunts hepatic fibrosis. Because endotoxin is a key activator of hepatic macrophages, this study was designed to test the hypothesis that LPS signaling through CD14 contributes to hepatic fibrosis caused by experimental cholestasis. Wild-type mice and CD14 knockout mice (CD14(-/-)) underwent sham operation or bile duct ligation and were killed 3 wk later. Measures of liver injury, such as focal necrosis, biliary cell proliferation, and inflammatory cell influx, were not significantly different among the strains 3 wk after bile duct ligation. Markers of liver fibrosis such as Sirius red staining, liver hydroxyproline, and alpha-smooth muscle actin expression were blunted in CD14(-/-) mice compared with wild-type mice after bile duct ligation. Despite no difference in lymphocyte infiltration, the macrophage/monocyte activation marker OX42 (CD11b) and the oxidative stress/lipid peroxidation marker 4-hydroxynonenal were significantly upregulated in wild-type mice after bile duct ligation but not in CD14(-/-) mice. Increased profibrogenic cytokine mRNA expression in the liver after bile duct ligation was significantly blunted in CD14(-/-) mice compared with the wild type. The hypothesis that LPS was involved in experimental cholestatic liver fibrosis was tested using mice deficient in LPS-binding protein (LBP(-/-)). LBP(-/-) mice had less liver injury and fibrosis (Siruis red staining and hydroxyproline content) compared with wild-type mice after bile duct ligation. In conclusion, these data demonstrate that endotoxin in a CD14-dependent manner exacerbates hepatic fibrogenesis and macrophage activation to produce oxidants and cytokines after bile duct ligation.

TNF alpha-induced Ras activation due to ethanol promotes hepatocyte proliferation independently of liver injury in the mouse.

Tumor necrosis factor alpha (TNFalpha) has been shown to be both proapoptotic and mitogenic for hepatocytes and necessary for alcohol-induced liver injury. Ras, a known proto-oncogene, is very important in the regulation of cellular responses to TNFalpha. Therefore, the purpose of this study was to investigate the role of Ras in alcohol-induced pathogenesis. Male C57Bl/6 mice were fed ethanol or high-fat control diet via intragastric cannulation for 4 weeks. Ras activity was increased significantly after 4 weeks of ethanol and correlated with an increase in pathologic features. However, in mice deficient in the receptor-type 1 for TNFalpha (TNFR1(-/-)), ethanol-induced liver injury and the increase in Ras activity were significantly blunted compared with wild-type mice. Furthermore, it was demonstrated that H-, K-, and R-Ras isoforms were increased after ethanol exposure in wild-type mice. In TNFR1(-/-) mice, R-Ras activity remained elevated by ethanol, whereas H-Ras and K-Ras activity was blunted significantly under these conditions. Interestingly, hepatocellular proliferation, which was elevated approximately fivefold after 4 weeks of chronic ethanol in wild-type mice, was also blunted in TNFR1(-/-) mice given ethanol. Inhibition of Ras with adenovirus containing a dominant-negative Ras had no effect on ethanol-induced liver injury, but significantly blunted ethanol-induced hepatocyte proliferation by more than 50%. Overexpression of mitochondrial superoxide dismutase using recombinant adenovirus blunted lipid peroxidation and attenuated hepatic injury resulting from ethanol, but had no effect on Ras activation and hepatocyte proliferation caused by ethanol. In conclusion, these data support the hypotheses that hepatocellular oxidative stress leads to cell death and that TNFalpha-induced Ras activation is important in hepatic proliferation in response to ethanol-induced liver injury.