Fig 1.
LCA inhibits IFNγ gene expression in Jurkat T cells
(A) 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) taurine-conjugated bile acids for 24 hours. (C) 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.
Fig 2.
LCA inhibits CD4+ Th cell activation.
(A) IFNγ mRNA expression and (B) TNFα 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). (C) IFNγ and (D) TNFα mRNA expression in P/I-activated Jurkat T cells in response to increasing concentrations of LCA. (E) 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). (F) mRNA expression of IFNγ, IL-6, CD40L, TNFα in primary mouse CD4+ Th cells activated with P/I and treated with 10 μμM LCA (light grey bars) or vehicle (dark grey bars). (G) mRNA expression of IFNγ, IL-6, CD40L, TNFα 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.
Fig 3.
LCA inhibits ERK phosphorylation in Jurkat T cells.
(A) Western blot images and (B) Quantification results of total and phosphorylated levels of ERK1/2, P38 and JNK1/2 in response to 10 μM LCA (light grey bars) or vehicle treatment (dark grey bars) in P/I-activated or resting Jurkat T cells. (C) Western blot images and (D) Quantification results of ERK1/2 phosphorylation in response to 10 μM LCA (light grey lines) or vehicle (dark grey lines) treatment in P/I-activated Jurkat T cells. LCA, lithocholic acid; P/I, PMA/ionomycin; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.
Fig 4.
LCA inhibits the Th1 differentiation response of CD4+ Th cells.
(A) mRNA expression in P/I-activated Jurkat T cells and (B) Dynabeads T-activator stimulated primary human CD4+ Th cells of a panel of genes associated with Th differentiation in response to 10 μM LCA treatment. ND; not detected. (C) Western blot images and (D) Quantification results of phosphorylated STAT1α/β and α-Tubulin in Jurkat T cells in response to P/I stimulation in combination with 10 μM LCA (light grey bars) or vehicle treatment (dark grey bars). LCA, lithocholic acid; P/I, PMA/ionomycin; ND, Not detected; AU, Arbitrary units. Results represent the mean ± SEM. *Statistically significant, P<0.05. Experiments were performed in triplicates and repeated at least twice.
Fig 5.
Characterizing the LCA sensor in CD4+ Th cells.
(A) 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) mRNA expression of TGR5, FPR3, S1PR2 PXR and VDR in resting “C” and in Dynabeads T-activator stimulated primary human CD4+ Th cells “A”. (C) 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). (D) IFNγ and (E) T-BET 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.