FXR activation enhances ACE2 expression and inhibits viral-induced IL-6 release from colonic epithelial cells in vitro: implications for gastrointestinal symptoms of SARS-CoV-2

Abstract

Introduction: While better known for its pulmonary symptoms, SARS-CoV-2 also adversely affects the gastrointestinal tract, causing diarrhoea with evidence of inflammation. The nuclear bile acid receptor, farnesoid x receptor (FXR), is expressed in colonic epithelial cells and has been previously shown to inhibit cytokine production and promote barrier function, thereby preventing inflammatory responses in the gut. In the current studies, we set out to investigate a potential role for FXR in modulating epithelial responses to SARS-CoV-2. Methods: Undifferentiated human colonic or ileal enteroids, colonic epithelial T84, or Caco-2 cells were all grown as monolayers on permeable supports. Cells were treated with the FXR agonists, obeticholic acid (OCA, 10 μM) or GW4064 (5 μM), or infected with live SARS-CoV-2 (2019-nCoV/USA_WA1/2020). Changes in cellular mRNA, protein or secreted cytokines were measured by qPCR, western blotting, or ELISA. SARS-CoV-2 levels were quantified by qPCR using primers for envelope (E) and nucleocapsid (N) protein. Data are expressed as mean ± SEM and statistical analysis was performed using paired t-tests or one-way ANOVA with Dunnett’s or Tukey’s multiple comparisons tests. Results: Treatment of colonic or ileal enteroids with the FXR agonist, OCA, increased expression of mRNA for the SARS-CoV-2 receptor, ACE2, by 2.1 ± 0.4 (n = 3; p = 0.08) and 2.3 ± 0.2 (n = 3; p < 0.05) fold, respectively. Similarly, treatment of T84 cells with another FXR agonist, GW4064, increased expression of ACE2 mRNA with a maximal response of 1.8 ± 0.2 fold (n = 8; p < 0.01) occurring after 72 hrs. Increased mRNA was accompanied by a 2.3 ± 0.7 fold (n = 6; p < 0.01) increase in cellular protein expression and a 1.4 ± 0.1 fold (n = 9; p < 0.001) increase in ACE2 protein secretion into the apical medium. Effects of FXR on ACE2 expression were confirmed in monolayers of Caco-2 cells where GW4064 induced a 1.5 ± 0.1 fold (n = 3; p < 0.001) increase in mRNA for the receptor. GW4064 treatment did not affect mRNA levels for SARS-CoV-2 E or N protein in the infected Caco-2 cells. However, further experiments revealed that treatment with GW4064 inhibited release of the proinflammatory cytokine, IL-6, from either Caco-2 cells infected with SARS-CoV-2 or from T84 cells treated with the viral mimic, polyinosinic-polycytidylic acid (poly (I:C)) (25 μg/ml) by 46 ± 12 % (n = 3, p < 0.05) and 35 ± 6 % (n = 8; p < 0.01), respectively. Conclusion: Our data suggest that FXR activation upregulates expression of ACE2 in intestinal and colonic epithelial cells but did not directly affect SARS-CoV-2 cellular levels. Rather, FXR activation inhibits viral-induced proinflammatory cytokine secretion and may therefore represent a good target for the development of new approaches to alleviate inflammatory diarrhoea associated with SARS-CoV-2 infection. This work was funded by grants from Science Foundation Ireland and the National Institutes of Health (DK047987). 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.