Farnesoid X receptor inhibits proinflammatory cytokine-induced epithelial necroptosis in vitro: implications for preservation of intestinal barrier function

Abstract

Epithelial cell death and compromised barrier function are key features of inflammatory bowel disease pathogenesis. Previous studies suggest that the nuclear bile acid receptor, farnesoid X receptor (FXR), promotes intestinal barrier function and protects against inflammation. Here, we investigated potential mechanisms involved. T₈₄ cell monolayers were treated with a combination of IFNγ and TNFα to model cytokine-induced barrier dysfunction in vitro. Apoptosis and necroptosis were assessed by measuring caspase 3/PARP cleavage and RIP3 phosphorylation, respectively. Epithelial permeability was determined by measuring 4-kDa fluorescein isothiocyanate-dextran (FD4) flux. Effects of FXR on barrier function in dextran sulfate sodium (DSS)-treated mice were assessed by measuring plasma levels of orally administered FD4. Treatment with IFNγ and TNFα enhanced FD4 flux and increased apoptosis in T₈₄ monolayers, as evidenced by increased cleaved PARP and caspase 3 levels. Pretreatment with the FXR agonist, GW4064, significantly inhibited cytokine-induced FD4 flux, but not apoptosis. Treatment with IFNγ and TNFα in the presence of the apoptosis inhibitor, Q-VD-OPh, induced necroptosis, as evidenced by increased RIP3 phosphorylation and enhanced FD4 flux, whereas a necroptosis inhibitor, necrostatin, inhibited these effects. GW4064 also inhibited cytokine-induced RIP3 phosphorylation and FD4 flux in the presence of Q-VD-OPh. In mice, treatment with the FXR agonist, obeticholic acid, attenuated DSS-induced disease activity and mucosal FD4 flux, but not levels of cleaved caspase 3 or phospho-RIP3. FXR activation inhibits cytokine-induced barrier dysfunction by inhibiting epithelial necroptosis rather than apoptosis in vitro. How such effects contribute to the protective actions of FXR in vivo requires further elucidation.NEW & NOTEWORTHY These studies demonstrate for the first time that FXR activation inhibits cytokine-induced necroptosis in vitro, an effect that may underlie protection against dysregulated barrier function in the setting of intestinal inflammation. These data support the potential for targeting FXR to promote epithelial barrier function in treatment of IBD.

Keywords: bile acid; farnesoid X receptor; inflammatory bowel disease; intestinal epithelium; necroptosis.