Extended Data Fig. 3: IsoLCA neither affects T cell viability nor inhibits Treg and TH1 cell differentiation in vitro.
From: Human gut bacteria produce ΤΗ17-modulating bile acid metabolites
a-c, IsoLCA does not reduce T cell viability or proliferation. Percentages of TH17 cells (a), viable cells (b) and total cell numbers (c) at the end of T cell culture under TH17 polarization conditions in the presence of LCA, 3-oxoLCA, or isoLCA at 40, 20, 10, 5, 2.5, 1.25 and 0.625 μM (n = 3 biologically independent samples, data are mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons). d–g, IsoLCA does not affect Treg or TH1 cell differentiation in vitro. Flow cytometry and quantification of intracellular staining for FoxP3 (d, e) or IFN-γ (f, g). Mouse naive CD4 T cells from wild-type C57BL/6J mice were cultured under TH1- or Treg- polarizing conditions and DMSO or isoLCA was added 18 h after TCR activation (n = 3 biologically independent samples per condition, data are mean ± SEM, two-tailed unpaired t-test). h, SFB colonization measured by qPCR analysis in Fig. 2c–f, calculated as SFB 16s rRNA copy number (n = 8 mice per group, pooled from two experiments, data are mean ± SEM, two-tailed unpaired t-test). i–k, Experimental scheme of Th17 induction by SFB (i), representative FACS plots (j) and population frequencies of TH17 cells (k), isolated from the ileal lamina propria of control or isoLCA-treated mice (n = 8 mice for control, n = 6 mice for isoLCA-treated groups, pooled from two experiments). B6 Tac mice were fed a control or an isoLCA (0.3% w/w)-containing diet for 7 days (data are mean ± SEM, two-tailed unpaired t-test). l–o, Experimental scheme of anti-CD3 experiment (l), representative FACS plots (m) and population frequencies of TH17 (n) and Treg cells (o) of the ileal lamina propria of control or isoLCA-treated mice (n = 15 mice for control, 13 mice for isoLCA-treated groups, pooled from three experiments). B6 Tac mice were intraperitoneally injected with anti-CD3 and fed a control diet or an isoLCA-containing (0.3% w/w) diet during the experiments (data are mean ± SEM, two-tailed unpaired t-test). p, RORγt luciferase reporter assay in HEK293 cells, treated with a synthetic RORγ inhibitor ML209 (1 μM), isoLCA (20 μM, 10 μM, 5 μM), isoDCA (20 μM, 10 μM, 5 μM) or DMSO. The fold ratio of firefly luciferase (FLuc) to Renilla luciferase (RLuc) activity is presented on the y-axis. DMSO-treated group set to 1 (n = 7 independent transfections per group, pooled from two experiments. Data are mean ± SEM, one-way ANOVA with Dunnett’s multiple comparison test, vehicle set as control). q, r, Differential scanning fluorimetry (DSF) analyses indicated robust binding of isoLCA (q), but not of isoDCA (r) to the RORγt ligand-binding domain (LBD). s–v, Surface plasmon resonance (SPR) indicated robust binding of isoLCA to the RORγt LBD. Sensorgrams for affinity (s) and kinetics (t) of isoLCA and affinity (u) and kinetics (v) of isoDCA with the RORγt LBD. w, Transcriptional profiling of wild-type (WT) T cells and RORγ deficient (KO) T cells, cultured under TH17 cell polarization conditions. DMSO or BAs were added to cells 18 h after TCR activation. Cells were then harvested, and RNA-sequencing was performed. Heat map represents 46 genes that are regulated by either 3-oxoLCA or isoLCA as well as RORγ (n = 3 mice per condition, the Wald test with Benjamini-Hochberg correction was used to determine FDR-adjusted p value < 0.05, genes that were differentially expressed by both isoLCA and 3-oxoLCA are shown in magenta). x, Gene ontology enrichment analysis was performed on the 46 genes that were differentially regulated by either 3-oxoLCA or isoLCA and RORγt revealed that these BA treatments resulted in changes in the expression of genes involved in several biological processes, including IL-17-mediated signalling and cytokine production pathways.
