LCA inhibits IFNγ gene expression in Jurkat T cells

<p><b>(A)</b> Cell viability of Jurkat T cells measured by flowcytometry analysis and expressed as percentage of control in response to incubation with unconjugated bile acids and <b>(B)</b> taurine-conjugated bile acids for 24 hours. <b>(C)</b> IFNγ mRNA expression of PMA/Ionomycin (P/I)-activated Jurkat T cells treated for 24 hours with 10 μM of different bile acid species. LCA, lithocholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid; TLCA, taurolithocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TCA, taurocholic acid; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.</p

Characterizing the LCA sensor in CD4⁺ Th cells.

<p><b>(A)</b> Table showing mRNA expression of Membrane and Nuclear bile acid receptors in Jurkat T cells and primary human CD4⁺ Th cells. “+” indicates that the gene is expressed at low levels, i.e. Real-time RT-PCR Ct cycles of 30–35; “++” indicates that the gene is expressed at Ct cycles of 25–30, and “+++” indicates high expression of the gene at Ct cycles of <25. <b>(B)</b> mRNA expression of <i>TGR5</i>, <i>FPR3</i>, <i>S1PR2 PXR</i> and <i>VDR</i> in resting “C” and in Dynabeads T-activator stimulated primary human CD4⁺ Th cells “A”. <b>(C)</b> mRNA expression of VDR in Jurkat T cells transfected with silencing RNA against the VDR (siVDR; light grey bar) or scrambled siRNA (siSCR; dark grey bar). <b>(D)</b> <i>IFN</i>γ and <b>(E)</b> <i>T-BET</i> mRNA expression in Jurkat T cells transfected with siRNA against the VDR (siVDR; light grey bars) or scrambled siRNA (siScr; dark grey bars) in response to P/I stimulation and in combination with 10 μM LCA or vehicle treatment. LCA, lithocholic acid; P/I, PMA/ionomycin; AU, Arbitrary units. Results represent the mean ± SEM. NS, Not significant. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.</p

LCA inhibits CD4⁺ Th cell activation.

<p><b>(A)</b><i>IFN</i>γ mRNA expression and <b>(B)</b> <i>TNF</i>α mRNA expression in Jurkat T cells in response to 10 μM LCA treatment (light grey lines) or vehicle (dark grey lines) with P/I activation (triangles) or in resting Jurkat T cells (squares). <b>(C)</b> <i>IFN</i>γ and <b>(D)</b> <i>TNF</i>α mRNA expression in P/I-activated Jurkat T cells in response to increasing concentrations of LCA. <b>(E)</b> Secreted TNFα protein levels of Jurkat T cells in the supernatant in response to 10 μM LCA treatment (light grey lines) or vehicle (dark grey lines) with P/I activation (triangles) or in resting Jurkat T cells (squares). <b>(F)</b> mRNA expression of <i>IFN</i>γ, <i>IL-6</i>, <i>CD40L</i>, <i>TNF</i>α in primary mouse CD4⁺ Th cells activated with P/I and treated with 10 μμM LCA (light grey bars) or vehicle (dark grey bars). <b>(G)</b> mRNA expression of <i>IFN</i>γ, <i>IL-6</i>, <i>CD40L</i>, <i>TNF</i>α in primary human CD4⁺ Th cells stimulated with Dynabeads T-activator (T-act) and treated with 10 μM LCA (light grey bars) or vehicle (dark grey bars). P/I, PMA/ionomycin; LCA, lithocholic acid; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.</p