Farnesoid X receptor activation attenuates intestinal inflammation and preserves epithelial barrier function in vivo and in vitro
Authors: Curley Caitriona , Lajczak-McGinley Natalia , Tambuwala Murtaza , Adorini Luciano , Fallon Ciara , Smyth Jessica , Keely Stephen 
Published: May 2023

Background: Increased epithelial cell death leading to compromised intestinal barrier function is a key contributor to the pathogenesis of inflammatory bowel disease. Previously published studies suggest that the nuclear bile acid receptor, farnesoid X receptor (FXR), promotes intestinal barrier function and is protective against colonic inflammation. Here, we investigated potential mechanisms involved. Methods: Mucosal inflammation was induced in mice by adding 2.5% dextran sulfate sodium (DSS) to their drinking water, either with or without daily oral gavage with the FXR agonist, obeticholic acid (OCA; 10 mg/kg). After 6 days, mice were administered FITC‐dextran (6 mg/kg) and then sacrificed 24hrs later. The severity of colonic mucosal inflammation was assessed by disease activity index (DAI) and mucosal permeability to FITC. Epithelial apoptosis was assessed by immunohistochemical imaging of cleaved caspase 3. To model the effects of cytokine-induced barrier dysfunction in vitro, we employed polarized monolayers of T84 colonic epithelial cells. Results were expressed as mean ± SEM and data were analyzed by one-way ANOVA, two-way ANOVA, and the Tukey’s post hoc test. Results: In mice, OCA treatment decreased the DSS-induced DAI score from 11.8 to 9.0 ± 0.7 (*p ≤ 0.05, n=6), increases in mucosal FITC flux by 62.3 ± 0.4% (*p ≤ 0.05, n=6), and epithelial caspase 3 cleavage by 68.1 ± 8.5% (n=6). In vitro studies revealed that treatment of T84 cells with the pro-inflammatory cytokines, IFNγ (10ng/ml) and TNFα (10ng/ml), induced apoptosis, as evidenced by increased levels of the apoptotic markers, cleaved PARP and cleaved caspase 3. However, pre-treatment of the cells with the FXR agonist, GW4064 (5μM), did not prevent cytokine-induced apoptosis. Treatment of T84 cells with IFNγ, TNFα and the apoptosis inhibitor, Q-VD-OPh, induced a necroptotic response, as evidenced by increased levels of phosphorylated RIP3 (pRIP3). Co-treatment with Q-VD-OPh also enhanced cytokine-induced transepithelial FITC flux to 3.0 ± 0.2 fold of that in control cells, whereas pre-treatment with the necroptosis inhibitor, necrostatin (200μM), reduced pRIP3 expression by 53.2 ± 3.4% (n=3) and FITC flux by 30.9 ± 0.2% (**p ≤ 0.01, n=10) of that in cells treated with Q-VD-OPh/IFNγ/TNFα alone. Similar to necrostatin, FXR activation with GW4064 inhibited both pRIP3 expression and FITC flux by 47.5 ± 9.1% (n=4) and 46.7 ± 0.6% (**p≤0.01, n=6), respectively, in this in vitro model of cytokine-induced necroptosis. Conclusion: Our studies show that FXR activation protects against intestinal inflammation, an effect that is likely due to preservation of epithelial barrier function. The protective effects of FXR activation on epithelial barrier function may be due to inhibition of necroptosis rather than apoptosis. Our data suggest that FXR represents a promising target for the development of new approaches to prevent epithelial barrier dysfunction in conditions of intestinal inflammation. Science Foundation Ireland (SFI) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.