Fig 6. Gut microenvironment and bile salts can drive EAE recovery and T cell apoptosis.
(A) Schematic representation of experiments in B and C. In vitro derived TH17 cells from C57BL6/J mice were treated with a metabolite in addition to mild anti-CD3 stimulation for 24 h. (B) Taurocholic acid (10 μm and 100 μm) was administered during anti-CD3 stimulation and apoptosis of TH17 cells isolated from C57BL6/J mice was measured using Annexin V staining by flow cytometry. (n = 5–6 mice/group; N = 2 experiments; mixed effects analysis with Geisser–Greenhouse correction [p = 0.0070]; Dunnett’s multiple comparison test [Veh vs. 10 μm p = 0.8178, Veh vs. 100 μm p = 0.0233]). (C) Administration of isovaleric acid and hexanoic acid (SCFAs that are increased in mice that recover) are sufficient to drive apoptosis in TH17 cells isolated from C57BL6/J mice. (n = 8 mice/group; N = 2 experiments; RM one-way ANOVA with the Geisser–Greenhouse correction [p = 0.0399]; Dunnett’s multiple comparisons test [Veh vs. 1 mM hexanoic p = 0.0764, Veh vs. 10 mM hexanoic p = 0.0352, Veh vs. 1 mM isovaleric p = 0.0026, Veh vs. 10 mM isovaleric p = 0.0254]). (D) Schematic depicting approach to FMT. (E) EAE clinical scores of C57BL6/J mice receiving FMT from separately housed Ahrfl/fl or Cd4creAhrfl/fl mice. (Females; representative plot includes n = 11 mice/group; total replicates of N = 2 experiments; Mann–Whitney U test on total scores reported in legend [p = 0.0096]). (F) Incidence of clinical sign development in mice treated with FMT. (G) Schematic depicting approach to oral supplement of 12.5 mg taurocholic acid/day in C57BL6/J mice immunized for EAE. (H) EAE clinical scores of C57BL6/J mice receiving 7 days of oral taurocholic acid (Females; n = 12 mice/group; total replicates of N = 1 experiment; Mann–Whitney U test on single days reported on plot). (I) Incidence of clinical sign development in mice treated with taurocholic acid. Error bars represent standard error from the mean. Raw data can be found in Supporting information (S1 Data). EAE, experimental autoimmune encephalomyelitis; FMT, fecal material transfer; SCFA, short-chain fatty acid.