Figure 5. Microbial metabolites of tryptophan and IFN-β suppress CNS inflammation via AhR in astrocytes.
(a) qPCR from sorted astrocytes and splenic DCs, macrophages or T cells from naive WT mice treated intranasally with 5.000 IU hIFN-β or PBS daily for 2 days (n = 3, Student’s t-test; normalized to Astrocytes Ahr). (b) EAE in control or GFAP-AhR mice under intranasal IFN-β treatment. Clinical scores of control (left) or GFAP-AhR-deficient (right) mice (mean ± s.e.m. in left graph; representative out of three independent experiments with n = 10 mice per group; Two-way ANOVA). (c) Left panel: RNA abundances from Control and GFAP-AhR astrocytes of indicated genes (n = 3, representative of two independent experiments, one-way ANOVA, Tukey’s multiple comparison test; normalized to Control Veh Vim). Middle graph: Quantification of CNS infiltrating CD11b+Ly6Chi inflammatory monocytes; representative out of three independent experiments with n = 10 mice per group; one-way ANOVA, Tukey’s multiple comparison test. Right graph: RNA analysis of pro-inflammatory gene cluster from sorted monocytes; ratio of count numbers of specific treatment group to Control veh; representative out of two independent experiments of pooled monocytes of n = 3 mice per group; one-way ANOVA, Tukey’s multiple comparison test. (d) GFAP-AhR-deficient and control animals under indicated treatment starting from day 22 after EAE induction (TDD: Tryptophan depleted diet; Trp: Tryptophan; Clinical scores, mean ± s.e.m.; representative out of two independent experiments with n = 10 mice per group; Two-way ANOVA; Tukey’s multiple comparison test). (e) qPCR of Ccl2 and Nos2 in indicated treatment conditions as in (c; normalized to Control TDD+Trp Ccl2, one-way ANOVA, Tukey’s multiple comparison test) (f, g, i) Clinical scores of WT mice treated with indicated conditions starting from day 22 after EAE induction (mean ± s.e.m.; representative out of two independent experiments with n = 5 mice per group; Two-way ANOVA; Tukey’s multiple comparisons test) (h) Left: qPCR of relative mRNA abundances for Ccl2 and Nos2 from astrocytes sorted at day 36 from experimental groups as in f, g, and i; (n = 3, representative of two independent experiments; one-way ANOVA followed by Tukey’s multiple comparisons test normalized to Vehicle Ccl2). Right: Measurement of I3S in urine samples collected at day 36 of experimental groups as in f, g, i (n = 3; representative of two independent experiments; one-way ANOVA followed by Tukey’s multiple comparison test). (j) SYBR Green qPCR of Lactobacillus reuteri bacterial DNA isolated from fecal samples of indicated groups at day 36 after EAE induction (n = 4 per group, representative of two independent experiments; one-way ANOVA followed by Tukey’s multiple comparisons test normalized to TDD+Trp). Significance levels: * P<0.05, ** P<0.01, *** P<0.001.