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.

Background Hepatosteatosis is associated with increased expression of tumor necrosis factor alpha (TNFα) and interleukin (IL)-12, major T helper (Th) 1 cytokines, and reduced hepatic 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. Methods Male C57Bl/6 wild type (Wt) and IL-12-deficient (IL12−/−) mice were fed a choline deficient diet (CDD) for 0, 10 or 20 weeks. Findings 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 (IFNγ) and TNFα in wt mice. Absence of IL-12 resulted in similar CDD-induced hepatosteatosis, but preserved hepatic NKT cells and significantly reduced hepatic IFNγ and TNFα expression. Treatment of CDD fed mice with lipopolysaccharide led to a significant increase in hepatic IL12 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 IL12 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 and NK cells. Conclusions Hepatosteatosis reduces the numbers of hepatic NKT cells in a KC and IL-12-dependent manner. Our results suggest a pivotal and multi-functional role of KC-derived IL-12 in the altered immune response in steatotic liver, a process which is likely active within human non-alcoholic fatty liver disease.

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.

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 cholinedeficient 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 nickend 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-␣), interleukin 12 (IL-12), and interferon gamma (IFN-␥) 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.)

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