Cholestatic liver disease occurs when bile flow is disrupted, intrahepatically and/or extrahepatically, leading to neutrophil accumulation and elevated bile acid concentrations especially taurine-conjugated bile acids. The robust neutrophilic inflammatory response propagates hepatocellular injury, which is dependent upon macrophage inflammatory protein-2 (MIP-2) expression. Previously, we demonstrated that taurocholic acid (TCA) induces the expression of MIP-2 via ERK activation of early growth response factor-1 (Egr-1) in hepatocytes. However, the mechanisms of bile acid induced neutrophilic inflammation are still not well understood.Interleukin-17A (IL-17A) is a Th17 cytokine that regulates neutrophil recruitment, defense against bacterial infection, and inflammation induced injury in autoimmune diseases. IL-17A expression is increased in patients with primary biliary cirrhosis and murine models of liver disease. Therefore, we hypothesized that IL-17A promotes inflammation in cholestatic liver disease. To test this hypothesis, we performed bile duct ligation (BDL), a murine model of obstructive cholestasis, on mice treated with an anti-IL-17A antibody. The neutralization of IL-17A reduced alanine aminotransferase (ALT) activity and areas of necrosis in mice subjected to BDL. The expression of pro-inflammatory mediators and neutrophil cell count was decreased in anti-IL-17A BDL mice. Furthermore, IL-17A synergistically enhances MIP-2 induction by TCA in hepatocytes. IL-17F, another member of the IL-17 family, did not affect hepatocellular injury or inflammation in BDL mice. However, IL-17F had a similar synergistic interaction with TCA in vitro. These data demonstrate that IL-17A is the predominant Th17 cytokine that contributes to the pathogenesis of cholestasis.To determine the molecular mechanisms that underline this synergistic interaction, we investigated known signaling pathways (i.e. C/EBPβ, p38, and JNK) that regulate IL-17A synergistic enhancement of pro-inflammatory mediators by other cytokines. The knockdown of C/EBPβ did not reduce IL-17A induction of MIP-2, but the upregulation of MIP-2 mRNA levels was attenuated in TCA treated hepatocytes. However, hepatocellular injury and inflammation was unaffected in C/EBPβ heterozygous knockout mice subjected to BDL for 3 days. Both p38 and JNK signaling pathways are activated by bile acids; whereas, treatment with IL-17A inhibited the activation of p38 and JNK. Consistently, inhibition of p38 and JNK signaling further promoted MIP-2 induction by bile acids. These results demonstrate the transcription factor, C/EBPβ, as an alternative pathway by which bile acid regulate the inflammatory response in hepatocytes. Furthermore, these data suggest that IL-17A inhibition of p38 and JNK signaling promotes the synergistic enhancement of MIP-2 induction by bile acids. Interleukin-23 (IL-23) is critical for Th17 cellular expansion and secretion of IL-17A. Mice subjected to BDL had a biphasic induction of IL-23 mRNA levels similar to TCA serum concentrations. Therefore, we tested the hypothesis that bile acids regulate hepatic IL-23 expression. IL-23 protein expression was increased in mice fed a 0.3% cholic acid diet. Furthermore, IL-23 protein and mRNA levels were elevated in hepatocytes treated with TCA via a JNK- and AKT-dependent manner. These data suggest that pathophysiological concentrations of bile acids promote a positive feedback loop in the IL-23/IL-17A axis during cholestasis.Overall, these studies demonstrate the molecular mechanisms underlying bile acid induction of the hepatic Th17 inflammatory response in cholestatic liver disease.
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