Richard J. Milton

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.

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

Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNF‐α) 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 Th1‐type cytokines IL‐12, interferon gamma (IFN‐γ), and TNF‐α in WT mice. The absence of IL‐12 resulted in similar CDD‐induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFN‐γ and TNF‐α 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. (HEPATOLOGY 2010;51:130–141.)

Favored T helper 1 response in a mouse model of hepatosteatosis is associated with enhanced T cell-mediated hepatitis.

Steatohepatitis enhances the severity of liver injury caused by acute inflammation. The purpose of this study was to test the hypothesis that fatty liver due to chronic choline‐deficient diet exacerbates concanavalin A (ConA)‐induced liver hepatitis, which is predominantly facilitated by T cells. Male C57BL/6 mice were fed either control choline‐sufficient diet (CSD) or choline‐deficient diet (CDD) for 6 weeks before ConA administration. Mice were sacrificed 3, 9, and 24 hours after ConA injection. Liver injury measured by aspartate aminotransferase (AST), alanine aminotransferase (ALT), pathology, and terminal deoxynucleotidyl transferase‐mediated nick‐end labeling (TUNEL) staining was minimal in mice fed either diet before ConA exposure. However, ConA‐induced liver injury was significantly greater in CDD‐fed mice compared with control‐fed mice. Liver cytokines were assessed by quantitative real‐time polymerase chain reaction (PCR). The expression of T helper (Th) 1 cytokines tumor necrosis factor alpha (TNF‐α), interleukin 12 (IL‐12), and interferon gamma (IFN‐γ) were dramatically elevated after ConA in CDD‐fed mice compared with control‐fed mice. CDD also enhanced ConA‐induced STAT4 activation, but not STAT6. Notably, regulators of T‐cell differentiation were strongly shifted toward a predominant Th1 profile. T‐bet, regulator of the Th1 response, was up‐regulated in CDD‐fed mice, whereas Th2 regulator GATA‐3 was significantly suppressed in CDD‐fed mice after ConA. Moreover, the expression of suppressor of cytokine signaling (SOCS)‐1, SOCS‐3, and repressor of GATA‐3 (ROG) favored a predominant Th1 cytokine response in CDD‐fed mice. In conclusion, these data support the hypothesis that hepatosteatosis caused by CDD is associated with more severe ConA‐induced hepatitis due to a predominant shift toward Th1 response. (HEPATOLOGY 2006;44:216–227.)

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

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. (HEPATOLOGY 2007;46:229–241.)

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

Transforming growth factor beta (TGFβ) 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 TGFβ‐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 TGFβ signaling on proinflammatory cytokine production, eosinophil recruitment, and hepatocellular apoptosis. Interruption of this pathway could be beneficial clinically to limit acute fulminant liver pathologies. (HEPATOLOGY 2007.)

TNF α‐induced ras activation due to ethanol promotes hepatocyte proliferation independently of liver injury in the mouse

Tumor necrosis factor α (TNFα) 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 TNFα. 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 TNFα (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 TNFα‐induced Ras activation is important in hepatic proliferation in response to ethanol‐induced liver injury. (HEPATOLOGY 2004;39:721–731.)