Bacteroides uniformis-induced perturbations in colonic microbiota and bile acid levels inhibit TH17 differentiation and ameliorate colitis developments

Abstract

Inflammatory bowel disease (IBD) is associated with gut dysbiosis and can lead to colitis-associated malignancies. Bacteroides uniformis (Bu) regulates animal intestinal homeostasis; however, the mechanism by which it alleviates colitis in mice remains unknown. We investigated the effects of B. uniformis JCM5828 and its metabolites on female C57BL/6J mice with dextran sulfate sodium salt (DSS) induced colitis. Treatment with Bu considerably alleviated colitis progression and restored the mechanical and immune barrier protein expression. Additionally, Bu increased the abundance of the symbiotic bacteria Bifidobacterium and Lactobacillus vaginalis while decreasing that of pathogenic Escherichia-Shigella, and modulated intestinal bile acid metabolism. Bu largely regulated the expression of key regulatory proteins of the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways in colonic tissues and the differentiation of TH17 cells. However, Bu could not directly inhibit TH17 cell differentiation in vitro; it modulated the process in the lamina propria by participating in bile acid metabolism and regulating key metabolites (alpha-muricholic, hyodeoxycholic, and isolithocholic acid), thereby modulating the intestinal immune response. Our findings suggest that Bu or bile acid supplements are potential therapies for colitis and other diseases associated with intestinal barrier dysfunction.

Fig. 1. B. uniformis JCM5828 ameliorates DSS-induced colitis by protecting against intestinal barrier damage and tight junction disruption.

a Schematic of the experimental design. Mice (female, n = 16 per group) were given 3.0% DSS for 24 d and treated with PBS or B. uniformis JCM5828 from d 15 to d 24. The number of mice surviving in the Con group on the day of sampling (n = 8) defined the final biological replicate of each group. b Changes in body weights during the experiments (n = 8). c DAI scores during the experiments (n = 8). d Survival curve during the experiments (n = 16). e A representative image of colon tissue from each group was provided, and the colon length was recorded (n = 8). f The spleen weight of each group (n = 8). g The histological score of the DSS-induced colitis was evaluated. h Representative microscopic images of mouse colon tissue stained with H&E and PAS (Scale bars = 200 μm) and corresponding local high magnification images (Scale bars = 50 μm). i The goblet cell count of the DSS-induced colitis was evaluated (n = 5). j qPCR analysis showing the mRNA expression of Occludin, ZO-1, Claudin-1 in colon tissues (n = 8). **p < 0.01, ***p < 0.001. Data were analyzed using one-way ANOVA with Tukey’s test and expressed as the means ± SEM.

Fig. 2. B. uniformis JCM5828 reshapes the intestinal microbiota and regulates intestinal bile acid metabolism.

Samples of colonic content from mice that were euthanized after treatment or died naturally during treatment were collected for 16 S rRNA and metagenomic sequencing. a The α diversity based on Chao, Simpson, and Shannon indexes (female, n = 8 per group). Data were analyzed using Kruskal–Wallis with Tukey–Kramer’s test. b Principle coordinate analysis (PCoA) plot based on the ASV matrix in three groups. β-diversity was determined using Adonis with Bray–Curtis test. c The Bacteroides to Firmicutes (Bac/Fir) ratio in three groups. Data were analyzed using one-way ANOVA with Tukey’s test. d Bacterial taxa identified as differentially abundant between groups according to LEfSe. The bacterial taxa of the three groups were compared at the genus level. Data were analyzed using the one-against-all multi-group comparison strategy, with an LDA threshold set at >4.0. e Differentially enriched bacteria between Bu and Con groups. Blue dots represent bacteria with higher abundance in the samples of Bu group. Gray dots represent bacteria with no significant difference between the two groups. P values were adjusted by Benjamini–Hochberg (BH) method to control FDR. FDR-adjusted P < 0.05 was shown. f Copy number of Bu in mouse colonic contents. Data were analyzed using one-way ANOVA with Tukey’s test. g Colonic microbial co-occurrence network analysis based on core ASV. Spearman’s rank correlation coefficient >0.60; P value <0.05. Different colors represent different genera in the colon. The size of nodes is proportional to the relative abundance of the ASV. The red and green lines indicate positive and negative correlations between species, respectively. h The potential functional pathways of colonic content microbiota based on PICRUSt2. Data differences were assessed using Kruskal–Wallis with Tukey–Kramer’s test. i Radar plot of differential metabolites in Con vs. Bu groups. Grid lines represent log2 values of difference multiples. j The relative abundance of nine differential metabolites in three groups. Differential metabolites were defined as metabolites with a fold change ≥2 and ≤0.5. Data were analyzed using Kruskal–Wallis with Tukey–Kramer’s test. *p < 0.05, **p < 0.01, ***p < 0.001. Data were expressed as the means ± SEM.

Fig. 3. B. uniformis JCM5828 alters colonic transcriptome profile in mice with DSS-induced colitis.

a Volcano plots for the RNA-Seq analyses of Con vs. Bu group. The red and green dots represent upregulated and downregulated DEGs in the Bu group, respectively. b The heatmap of DEGs expression in two groups. c The KEGG enrichment analysis of Con vs. Bu group. The genes behind each bar represent the gene involved in the pathway. The red and green genes represent upregulated and downregulated DEGs in the Bu group, respectively. d Bu binds to the IL-17 receptor (IL-17R) in colon epithelial cells and inhibits downstream NF-κB and MAPK signaling pathways through the signaling complex IL-17R-ACT1-TRAF6, thereby eliminating the transcription and expression of inflammatory factors in the nucleus. The blue box represents KEGG annotated differential genes downregulated in the Bu group. e, f Western blot analyses of p-P38/P38, p-JNK1/2/JNK1/2, p-ERK1/2/ERK1/2, p-P65/P65, p-IκB/IκB, and p-IKK/IKK in colon tissues using the respective anti-phospho-protein antibodies, and quantified phosphorylated protein and total protein density (n = 8). Data were analyzed using one-way ANOVA with Tukey’s test and expressed as the means ± SEM. *p < 0.05, ***p < 0.001.

Fig. 4. B. uniformis JCM5828-mediated bile acids inhibit TH17 differentiation and alleviate DSS-induced colitis.

a Gating strategy for the flow cytometric sorting of intestinal CD4⁺ T cells. Flow cytometric analyses (b) and quantification (c) of RORγt production from C57BL/6J mice naive T cells cultured for 3 d under the TH17 cell polarization condition. PBS, PBS, or Bu supernatant (MOI = 1:10) were added after LPS addition (n = 3 respectively, biologically independent samples). d Gating strategy for the flow cytometric sorting of intestinal CD4⁺ T cells. Flow cytometric analyses (e) and quantification (f) of RORγt production from C57BL/6J mice naive T cells cultured for 3 d under the TH17 cell polarization condition. DMSO, Bu supernatant (MOI = 1:10), or bile acids (20 μM) were added after cytokine addition (n = 3 respectively, biologically independent samples). Data were analyzed using one-way ANOVA with Tukey’s test and expressed as the means ± SEM. ***p < 0.001.

Fig. 5. The mixed bile acids attenuated symptoms in the DSS-induced colitis mouse model.

a Schematic of the experimental design. Mice (female, n = 8 per group) were given 3.0% DSS for 14 d and treated with PBS, Bu, inactivated Bu, or mixed bile acids from d 7 to d 14. b Changes in body weights during the experiments. c DAI scores during the experiments. d A representative image of colon tissue from each group was provided, and the colon length was recorded (n = 8). e The spleen weight of each group (n = 8). f Representative microscopic image of H&E staining of mouse colon tissue (Scale bars = 200 μm) and corresponding local high magnification images (Scale bars = 20 μm). g Representative microscopic image of PAS staining of mouse colon tissue (Scale bars = 200 μm) and corresponding local high magnification images (Scale bars = 100 μm). Histological scores (h) and goblet cell count (i) of the DSS-induced colitis were evaluated (n = 8). j qPCR analysis showing the mRNA expression of IL-1β, IL-6, TNF-α, and IL17A in colon tissues (n = 8). Data were analyzed using one-way ANOVA with Tukey’s test and expressed as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. The mixed bile acids inhibits TH17 differentiation.

a Gating strategy for the flow cytometric sorting of intestinal CD4⁺ T cells. Flow cytometric analyses (b) and quantification (c) of Th17 (CD3⁺CD4⁺RORγt⁺) frequencies in the colonic lamina propria of C57BL/6J mice in each group (n = 3 respectively, biologically independent samples). Data were analyzed using one-way ANOVA with Tukey’s test and expressed as the means ± SEM. ***p < 0.001.

Fig. 7. The processes behind the protective effect of B. uniformis JCM5828 against DSS-induced colonic inflammation in mice are summarized in a schematic picture.

Bu gavage changed the colonic microbial composition of mice with DSS-induced colitis, and primary bile acids (α-MCA) synthesized by the Bu core functional enzyme gene BSH involved in enhancing intestinal barrier function and inhibiting TH17 cell differentiation along with other putative probiotic-mediated secondary bile acids. TH17 cells in the colon epithelium bind to the IL-17 receptor (IL-17R) and inhibit downstream NF-κB and MAPK signaling pathways via the signaling complex IL-17R-ACT1-TRAF6. This reduces the expression of inflammatory factors such as TNF-α, IL-1β, and IL-6, modulating the intestinal immune response. Figdraw software was used to create this illustration.