Hepatic NFAT signaling regulates the expression of inflammatory cytokines in cholestasis

Abstract

Background & aims: The nuclear factor of activated T-cells (NFAT) plays an important role in immune responses by regulating the expression of inflammatory genes. However, it is not known whether NFAT plays any role in the bile acid (BA)-induced hepatic inflammatory response. Thus, we aimed to examine the functional role of NFATc3 in cholestatic liver injury in mice and humans.

Methods: Gene and protein expression and cellular localization were assessed in primary hepatocyte cultures (mouse and human) and cholestatic liver tissues (murine models and patients with primary biliary cholangitis [PBC] or primary sclerosing cholangitis [PSC]) by quantitative PCR, western blot and immunohistochemistry. Specific NFAT inhibitors were used in vivo and in vitro. Gene reporter assays and ChIP-PCR were used to determine promoter activity.

Results: NFAT isoforms c1 and c3 were expressed in human and mouse hepatocytes. When treated with cholestatic levels of BAs, nuclear translocation of NFATc3 was increased in both human and mouse hepatocytes and was associated with elevated mRNA levels of IL-8, CXCL2, and CXCL10 in these cells. Blocking NFAT activation with pathway-specific inhibitors or knocking down Nfatc3 expression significantly decreased BA-driven induction of these cytokines in mouse hepatocytes. Nuclear expression of NFATc3/Nfatc3 protein was increased in cholestatic livers, both in mouse models (bile duct ligation or Abcb4^-/- mice) and in patients with PBC and PSC in association with elevated tissue levels of Cxcl2 (mice) or IL-8 (humans). Gene reporter assays and ChIP-PCR demonstrated that the NFAT response element in the IL-8 promoter played a key role in BA-induced human IL-8 expression. Finally, blocking NFAT activation in vivo in Abcb4^-/- mice reduced cholestatic liver injury.

Conclusions: NFAT plays an important role in BA-stimulated hepatic cytokine expression in cholestasis. Blocking hepatic NFAT activation may reduce cholestatic liver injury in humans.

Lay summary: Bile acid induces liver injury by stimulating the expression of inflammatory genes in hepatocytes through activation of the transcription factor NFAT. Blocking this activation in vitro (in hepatocyte cultures) and in vivo (in cholestatic mice) decreased the expression of inflammatory genes and reduced liver injury.

Keywords: Bile acids; Cholestatic liver injury; Inflammatory cytokines; Nuclear factor of activated T-cells.