Mechanisms of Proinflammatory Signaling during Cholestasis

Abstract

Cholestasis is a condition in which bile flow from the liver to the intestines is inhibited. Loss of bile flow leads to increased concentrations of bile constituents, including bilirubin and bile acids within the liver. Cholestasis leads to liver inflammation and injury. Several studies have demonstrated that inflammation is required for injury; however, the mechanism by which cholestasis stimulates proinflammatory gene expression remains unknown. We demonstrated previously that the transcription factor early growth response factor-1 (Egr-1) is critical for inflammation and injury during cholestasis, and that Egr-1 is upregulated in deoxycholic acid (DCA)-treated primary mouse hepatocytes. To determine the mechanism by which Egr-1 is upregulated during cholestasis we tested the hypothesis that activation of the farnesoid X receptor (FXR) or the mitogen-activated protein kinase (MAPK) pathway is required for upregulation of Egr-1 during cholestasis. The results demonstrated that loss of FXR signaling did not alter Egr-1 expression in hepatocytes treated with bile acids or during bile duct ligation (BDL), a model of cholestasis. However, loss of MAPK singling prevented upregulation of Egr-1 both in vitro and in vivo. Next, we tested the hypothesis that bile acids upregulate proinflammatory mediators in hepatocytes by Egr-1-dependent mechanisms. The results demonstrated that the bile acids DCA, chenodeoxycholic acid (CDCA) and taurocholic acid (TCA) increased proinflammatory gene expression in primary mouse hepatocytes by Egr-1-dependent and independent mechanisms. This study also demonstrated that upregulation of proinflammatory genes by bile acids occurred independently of cell death. Further studies demonstrated that upregulation of plasminogen activator inhibitor type-1(PAI-1), vascular cell adhesion molecule-1, and Ccl7 in BDL mice required Egr-1. Lastly, Egr-1 was upregulated in the livers of patients with cholestasis and correlated with levels of IL-8, intercellular adhesion molecule-1 and PAI-1. Next, we tested the hypothesis that interleukin-17 (IL-17) family members are required for inflammation during cholestasis. The IL-17 family of cytokines has been associated with neutrophilic inflammation and induction of proinflammatory gene expression in some cell types. Our results demonstrated that IL-17D, but no other IL-17, was upregulated in bile acid-treated hepatocytes. In addition, treatment of BDL mice with an anti-IL-17D antibody attenuated upregulation of some proinflammatory mediators, but did not affect liver injury or inflammation. Next we investigated the role of IL-17A and IL-17F in regulation of inflammation during cholestasis. IL-17A was inhibited with a neutralizing antibody, and IL-17A and IL-17F signaling were inhibited with a neutralizing antibody against IL-17 receptor A. BDL mice treated with anti-IL-17A antibody had similar liver injury and inflammation as control-treated mice. Similarly, treatment of mice with an anti-IL-17 receptor A antibody during cholestasis did not affect liver injury or inflammation. Overall the results from our studies identified novel mechanisms of inflammation during cholestasis, and reveal potential drug targets for the treatment of cholestatic liver diseases.