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.

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Transforming growth factor beta (TGF␤) 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 TGF␤-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 TGF␤ signaling on proinflammatory cytokine production, eosinophil recruitment, and hepatocellular apoptosis. Interruption of this pathway could be beneficial clinically to limit acute fulminant liver pathologies. ( The inherent ability of TGF␤ to regulate immune responses is strong evidence for it being an endogenous regulator of T cell function. 2 In fact, TGF␤ exerts great impact on T cells, as is evident in mice with a T cellspecific blockade of TGF␤ signaling, which resulted in the activation of a self-targeted immune response. 3,4 Additionally, recent studies in mice and humans have elucidated an important and complex role for TGF␤ in autoimmune diseases, 5 which are very often mediated by disrupted T cell signaling. 6 Inhibition of TGF␤ is associated with increased susceptibility to different experimental models of autoimmune diseases, including rheumatoid arthritis, insulin-dependent (type 1) diabetes mellitus, and inflammatory bowel disease. 7-9 Surprisingly, the role of TGF␤ in autoimmune hepatitis is not known. Recent work suggests that TGF␤ inhibits the development of immunopathology to self or otherwise nonharmful antigens without compromising immune responses to pathogens. This appears to be achieved by TGF␤-controlled initiation and resolution of inflammatory responses mediated by the regulation of chemotaxis, activation, and...

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TGFβ signaling through Smad3 plays a multifunctional role within the liver, promoting myofibroblast production of collagen and inducing hepatocellular apoptosis. The purpose of the current study was to evaluate the importance of Smad3 signaling during chronic cholestasis. Wild type (wt) or Smad3−/− mice were subjected to ligation of the common bile duct (BDL) or sham surgery and allowed to recover. Twenty one days following BDL, wt mice presented with significant increases in serum transaminases and hepatocellular apoptosis as assessed by terminal UTP nick end labeling (TUNEL) as well as significant myofibroblast activation and hepatic collagen deposition. Smad3−/− mice subjected to the same duration of cholestasis present with significant reductions in hepatocellular injury as assessed by serum aspartate aminotransferase levels and reduced hepatocellular apoptosis, findings which correlated with reduced collagen deposition but similar stellate cell activation. Together, these data demonstrate the importance of Smad3 signaling not only in the production of collagen by intrahepatic myofibroblasts but also in the induction of hepatocellular apoptosis following chronic cholestasis. Interruption of TGFβ signaling may therefore represent an important therapeutic target for the treatment of both hepatocellular injury and tissue fibrosis. This work was supported by NIH grant AA0014243.

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