Microbiota-produced immune regulatory bile acid metabolites control central nervous system autoimmunity

Abstract

The commensal gut microbiota has a role in the pathogenesis of extra-intestinal autoimmune diseases such as multiple sclerosis (MS) with unknown mechanisms. Deoxycholic acid (DCA) and lithocholic acid (LCA) are secondary bile acid metabolites (BAMs) produced from primary bile acids by gut microbiota that play key immune regulatory functions by promoting FOXP3⁺ regulatory T (Treg) cell differentiation at the expense of Th17 cells. Here, we show that bacteria releasing enzymes responsible for secondary BAMs production are under-represented in the gut of MS patients, resulting in significantly reduced intestinal concentration of DCA and immune dysregulation with increased percentage of Th17 cells. We validated our human findings in a preclinical model of MS by showing that DCA/LCA administration prevents experimental autoimmune encephalomyelitis (EAE) by dampening Th17 cell differentiation and the effector phenotype of myelin-reactive T cells. Our data highlight the key role of immune regulatory BAMs for the prevention of central nervous system (CNS) autoimmunity.

Keywords: FoxP3+ Treg cells; T helper 17 cells; bile acid metabolites; metabolomics; microbiome; multiple sclerosis.

Graphical abstract

Figure 1

Reduced representation of BAM-producing bacteria in the gut microbiome of RRMS patients (A) Schematic representation of the human study. (B) Violin plots showing alpha diversity in HCs (n = 20) and RRMS patients (n = 20) based on Shannon index. (C) MA plot showing the differential abundance of bacterial species significantly altered in HCs and RRMS patients (false discovery rate [FDR] < 0.1). (D) Frequency of reads related to BAM-producing bacteria in HCs and RRMS patients. (E) Heatmap showing the relative abundance of relevant BAM-producer bacterial species that were significantly reduced in RRMS patients (FDR < 0.1). See also Tables S1 and S2 and Figure S1A.

Figure 2

Low DCA concentrations in the intestine of RRMS correlate with disease occurrence (A) Boxplots of UPLC mass spectrometry peak intensity (logarithmic scale) showing the abundance of secondary BAMs detected in fecal samples of RRMS patients (n = 19) and HCs (n = 20). Data were generated upon Mann-Whitney U test among the two experimental sample groups. (B) Multivariate analysis showing correlation among the concentration of DCA, hepatic functions (AST, ALT, GGT, direct bilirubin, indirect bilirubin, and total bilirubin), clinical data (disease duration and EDSS), and potential confounders such as age and sex. Multivariate analysis was performed by Spearman correlation coefficients and adjusted by FDR. Correlation coefficients in yellow are significant at p < 0.05. (C) Bacterial species releasing enzymes performing 7-α-dehydroxylation that were significantly reduced in RRMS samples compared to HCs (FDR < 0.1). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. See also Figures S1B and S2.

Figure 3

Alteration of the microbiota-regulated metabolic profile is linked to immune dysregulation in RRMS (A) Percentage of circulating T cell subsets in RRMS patients and HCs measured by multiparametric flow cytometry. γδT cells were identified as CD45⁺CD3⁺TCRγδ⁺ cells; MAIT cells were defined as CD161⁺TCRVα7.2⁺ of CD3⁺TCRγδ⁻ cells. Among non-MAIT/non-TCRγδ CD4⁺ T cells, we identified Treg cells as CD25⁺FOXP3⁺ cells. Finally, the FOXP3-negative T cell population was further divided in the following effector T helper (Th) cells: Th1 as FOXP3⁻Tbet⁺, Th17 as FOXP3⁻RORγt⁺, and Th2 as FOXP3⁻CRTH2⁺. (B) In vitro expansion of Tbet⁺ Th1, RORγt⁺ Th17, FOXP3⁺ Treg cells and CRTH2⁺ Th2 cells in response to gut microbial filtrates of HCs and RRMS patients. PBMCs from a healthy donor were cultured for 72 h in the presence of fecal filtrates derived from HCs or RRMS patients and analyzed by flow cytometry as in (A). Each symbol represents a fecal sample donor (n = 10 per group). Data are expressed as fold difference over vehicle control (no fecal filtrates). (C) Percentage of in vitro expanded RORγt⁺ Th17 cells in response to gut microbial filtrates of RRMS patients supplemented or not with 10 μM DCA. PBMCs from a healthy donor were cultured for 72 h and subsequently analyzed by flow cytometry (n = 10 fecal sample donor per group). Bars represent the mean ± SD of data from two independent experiments. All statistical analyses were performed using Mann-Whitney U test to compare the RRMS group with the HC group or the RRMS fecal material (FM) group with the RRMS FM + DCA group. ∗p < 0.05, ∗∗p < 0.01; ns, not significant. See also Figures S3 and S8.

Figure 4

Supplementation with immune regulatory secondary bile acid metabolites (BAM) dampens CNS autoimmunity (A) Schematic illustration of EAE induction and DCA/LCA supplementation. (B) Clinical score and disease incidence in MOG_35-55 immunized mice following BAM supplementation (n = 10 mice per group); clinical score data are expressed as mean ± SEM. EAE clinical scores were analyzed with two-way ANOVA test followed by Bonferroni post-testing for multiple comparisons. Disease incidence was evaluated using the Kaplan-Meier estimation, whereas statistical significance was evaluated by the log rank (Mantel-Cox) test. (C) Histological analysis of the spinal cord of EAE-immunized mice receiving BAM or vehicle (n = 5 mice per group). Adjacent sections are stained to detect lymphocyte infiltrates (hematoxylin and eosin staining), demyelination (Kluver-Barrera staining), and axonal loss (Bielschowsky staining). Magnification 20×. Scale bar 200 μm. Bars represent the mean ± SD. Unpaired t test was used for statistical analysis. One representative experiment out of two is shown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. See also Figures S5 and S6.

Figure 5

Immune regulatory secondary bile acid metabolites (BAM) reduce inflammation and promote intestinal immune tolerance in EAE mice (A) Percentages of CD4⁺ T cells expressing IL-17A (Th17), IFN-γ (Th1), FOXP3 and CD25 (FOXP3⁺ Treg), or LAG3 and CD49b (FOXP3⁻ Tr1) in the intestine of EAE mice supplemented with BAM or vehicle (n = 5 mice per group). (B) percentages of total CD4⁺ T cells and absolute numbers of effector Th17, Th1, and regulatory FOXP3 and Tr1 cells in the intestinal mucosa of EAE-immunized mice (n = 5 mice per group), treated or not with BAM. Bars represent the mean ± SD. Unpaired t test was used. (C) RT-qPCR analysis of cytokine mRNA expression in the intestine of EAE-immunized mice at the time of onset of clinical signs of the disease and control (not immunized) mice receiving oral BAM supplementation or vehicle (n = 7–10 mice per group). Data are expressed as mean ± SD. One-way ANOVA was performed, followed by Tukey’s test. Data are from one representative experiment out of two. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. See also Figures S4, S7, and S9.

Figure 6

Immune regulatory secondary bile acid metabolites (BAM) modulate peripheral immunity in EAE mice (A) Percentages of CD4⁺ T cells expressing IL-17A (Th17), IFN-γ (Th1), FOXP3 and CD25 (FOXP3⁺ Treg), or LAG3 and CD49b (FOXP3^- Tr1) in the lymph nodes draining the immunization site (DLN) of EAE mice supplemented with BAM or vehicle. (B) Percentages of CD4⁺ α4β7⁺ T cells among Th17, Th1, FOXP3⁺ Treg, and FOXP3^- Tr1 cells in the DLN of BAM-treated EAE mice. (C) Percentages of Th17, GM-CSF⁺Th17, Th1, GM-CSF⁺ Th1, FOXP3⁺ Treg, and FOXP3^- Tr1 cells in the CNS of BAM-treated or vehicle-treated EAE-immunized mice. (D) Percentages of Th17, GM-CSF⁺Th17, Th1, and GM-CSF⁺ Th1 cells in cell cultures enriched for MOG-specific T cells obtained from lymphocytes isolated from DLN of EAE mice supplemented or not with BAM. Bars represent the mean ± SD. (n = 5 mice per group). Unpaired t test was performed. Data are from one representative experiment out of two. ∗p < 0.05, ∗∗p < 0.01; ns, not significant. See also Figure S9.

Figure 7

Immune regulatory DCA and LCA modulate the functional phenotype of myelin-specific T cells Bone marrow-derived dendritic cells (DCs) were pulsed with the MOG35-55 peptide; stimulated with LPS (1 μg/mL) alone or in the presence of DCA, LCA, or DCA/LCA; and co-cultured for 72 h with naive CD4⁺ T cells isolated from 2D2 TCR^MOG transgenic mice (DC:naive T cell ratio = 1:5, n = 3 per group). Representative flow cytometry plots (right) and bar graphs with individual values expressing the mean percentages ± SD (left) of Th17 (CD4⁺ IL-17A⁺), Th1 (CD4⁺ IFN-γ⁺), and GM-CSF⁺ Th17 and Th1 cells out of total CD4⁺ T cells from triplicate wells. One representative experiment out of two is shown. One-way ANOVA was used, followed by Dunnett’s test for multiple comparisons. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant.