Clostridium sporogenes-derived metabolites protect mice against colonic inflammation

Abstract

Gut microbiota-derived metabolites play a pivotal role in the maintenance of intestinal immune homeostasis. Here, we demonstrate that the human commensal Clostridium sporogenes possesses a specific metabolic fingerprint, consisting predominantly of the tryptophan catabolite indole-3-propionic acid (IPA), the branched-chain acids (BCFAs) isobutyrate and isovalerate and the short-chain fatty acids (SCFAs) acetate and propionate. Mono-colonization of germ-free mice with C. sporogenes (CS mice) affected colonic mucosal immune cell phenotypes, including up-regulation of Il22 gene expression, and increased abundance of transcriptionally active colonic tuft cells and Foxp3⁺ regulatory T cells (Tregs). In DSS-induced colitis, conventional mice suffered severe inflammation accompanied by loss of colonic crypts. These symptoms were absent in CS mice. In conventional, but not CS mice, bulk RNAseq analysis of the colon revealed an increase in inflammatory and Th17-related gene signatures. C. sporogenes-derived IPA reduced IL-17A protein expression by suppressing mTOR activity and by altering ribosome-related pathways in Th17 cells. Additionally, BCFAs and SCFAs generated by C. sporogenes enhanced the activity of Tregs and increased the production of IL-22, which led to protection from colitis. Collectively, we identified C. sporogenes as a therapeutically relevant probiotic bacterium that might be employed in patients with inflammatory bowel disease (IBD).

Keywords: Colitis; commensal bacteria; indole-3-propionate; microbial metabolites.

Figure 1.

C. sporogenes synthesizes a broad range of metabolites in the gut of mice. (a) Levels of acetate, propionate, butyrate (n = 4) and (b) numerous small polar molecules in the caecum of GF, OMM and SPF mice, measured by GC-MS and LC-MS. (c) Levels of tryptophan in the colon of GF, CS and SPF mice measured by GC-MS (n = 3). (d) Detection of indole-3-propionic-acid (IPA) in GF, CS, OMM and SPF mice in the ileum (n = 2), caecum, (n = 3) colon (n = 3) and serum (n ≥3). Pooled human serum samples were measured as a reference (n = 3). (e) Levels of SCFAs and (f) BCFAs in the colon of GF and CS mice measured by GC-MS (n = 4). Statistical analysis was done with one-way ANOVA with *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Data are presented as mean and error bars represent SD.

Figure 2.

Alterations in the local immune system and colonic tuft cell abundance in mice mono-colonized with C. sporogenes. (a) Representative flow cytometry dot-blots and scatter blot of lamina propria FoxP3⁺ CD4⁺ T cells in the colon of GF and C. sporogenes mono-colonized (CS) mice (n = 8). (b) Bar graphs, showing the relative fold change of Foxp3, Il13, and Il22 expression in the colon of GF, CS and SPF mice via quantitative real time PCR (qPCR). Expression is shown relative to hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression (n = 3). (c,d) flow cytometry analysis of Th17 cells showing the percentage of IL-22⁺ cells after 3 days. CD4⁺ T cells were isolated from two to four month-old C57BL/6N mice and differentiated under Th17-polarising conditions for 3 days. (c) Cells were treated with C. sporogenes metabolites (IPA 50 µm; propionate, isobutyrate, valerate, isovalerate, each 2 mm; butyrate 0.5 mm) or (d) bacterial cell-free supernatants from day 0 on. (n ≥3). Statistical analysis was done with one-way ANOVA with *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Data are presented as mean. (e) In double-labeling ISH experiments, colonic tuft cells (Egfp_dig-labeled in chat-egfp mice) were found Ffar2 negative, but Ffar3 co-positive (representative images of n ≥100 analyzed cells). (f) Representative detection of Pou2f3⁺ tuft cells (labeled by red arrows) by immunohistochemistry in the gut of mice. (g) The density of colonic epithelial tuft cells was analyzed in GF, CS, and SPF mice by Pou2f3 immunohistochemistry (n = 7). Each point represents the mean of 3 single measurements. (h) Pou2f32f3 immunoreactivity discriminated C1-type (cytoplasmic signals) from C2/3-type (nuclear signals) colonic tuft cells. (i) Quantification of C2/3-type tuft cells in the colon of GF, CS, and SPF mice (n = 4). Statistical analysis was done with two-way ANOVA with Tukey post hoc test with *p < 0.05, ****p < 0.0001.

Figure 3.

C. sporogenes protects mice against dss-induced colitis. (a) Survival, (b) weight curve and (c) histopathology scores of GF, CS and SPF mice treated with DSS (2.5%) in drinking water for 5 days (n ≥7). Statistical analysis was done with one-way ANOVA (a) and two-way ANOVA (c) with *p < 0.05, ****p < 0.0001. The horizontal line represents the mean. (d, e) Representative colon tissue sections from DSS-treated GF, CS and SPF mice stained with (d) hematoxylin and eosin (HE) or (e) Alcian blue and nuclear red (n = ≥ 7).

Figure 4.

Analysis of epithelial and T cells in the colon of GF, CS and SPF mice. (a-d) GF, CS and SPF animals were orally treated with DSS (2.5%) in drinking water for 5 days and analyzed on day 7. (a,b) Representative immunohistochemistry staining of colon tissue and scatter plot from DSS-treated GF, CS and SPF mice stained with (a) anti-Dclk1 or (b) anti-CD3 antibodies (n = 7). Statistical analysis was done with two-way ANOVA with Tukey post hoc test with ****p < 0.0001. The horizontal line represents the mean. (c) Scatter plots showing CD4⁺ cells in the lamina propria of the colon in DSS-treated GF, CS and SPF mice. Shown are the percentage and cell count of CD4⁺ cells, cell count of IL-17A⁺ CD4⁺ and IFNγ⁺ CD4⁺ cells and the percentage of FoxP3⁺ CD4⁺ cells (n = 4-5). Statistical analysis was done with one-way ANOVA with *p < 0.05; **p < 0.01. The horizontal line represents the mean. (d) Heat map displaying significantly changed genes related to epithelial cell function the between GF and CS mice after DSS-induced colitis. Colon tissue was harvested, and RNA was extracted and sequenced (n = 3). Expression changes of genes were considered significant with p adjusted value < 0.05. Transcripts per million (TPM) values were computed, z-score transformed and represented in heatmaps. Heatmaps were generated with the R package pheatmap v. 1.0.12 using Euclidean distances. Trees indicate hierarchical clustering (complete method).

Figure 5.

C. sporogenes influences Th17 cells in DSS-induced colitis. (a-c) GF, CS and SPF animals were orally treated with DSS (2.5%) in drinking water for 5 days and analyzed on day 7. Colon tissue was harvested, and RNA was extracted and sequenced (n = 3). Expression changes of genes were considered significant with p adjusted value < 0.05). (a) Venn diagram indicating the amount of significantly changed genes between the compared groups. Number of genes only changed in the comparison GF vs CS are red, SPF vs CS green and SPF vs GF blue. (b) Volcano plot of murine colon tissue after DSS treatment showing differently regulated genes between GF and CS mice. (c) KEGG pathway analysis showing the top 10 most changed pathways between SPF, CS, and GF mice.

Figure 6.

IPA reduces amounts of IL-17A in Th17 cells. (a-i) CD4⁺ T cells were isolated from two to four month-old C57BL/6N mice and differentiated under Th17-polarising conditions for 2 days for RNA-based and 3 days for protein-based methods. Cells were treated with different stimuli starting at day 0. (a) Flow cytometry analysis of Th17 cells showing percentage of IL-17A⁺ cells after 3 days (n = 3). (b) Flow cytometry analysis of Th17 cells showing the percentage of IL-17A⁺ cells after 3 days. Cells were treated with 2.5 vol% bacterial cell-free supernatant set to an OD₆₀₀ of 0.7 (n ≥3). (c) Staining of RORγt and IRF4 in Th17 cells via flow cytometry on day 3 (n = 3). (d) Heatmap displaying all significantly (padj. <0.05) changed genes between untreated and ipa-treated Th17 cells. Trees indicate hierarchical clustering (complete method). (e) KEGG pathway analysis showing the top 10 most changed pathways in ipa-treated Th17 cells compared to the control. (f) Phospho-staining of p-mTOR in Th17 cells via flow cytometry on day 3 of the cell culture (n ≥3). (g) Phospho-staining of p-S6 in Th17 cells via flow cytometry on day 3 of differentiation (n = 4). (h) Representative western blot of phosphorylated-4E-BP1 in Th17 cells and bar graphs showing relative p-4E-BP1 signals normalized to β-actin (n = 3). (i) Flow cytometry analysis of Th17 cells purified from WT (white) and Ahr^−/− (grey) mice showing the percentage of IL-17A⁺ cells after 3 days differentiation (n = 4). All statistical analyses were done with one-way ANOVA with *p < 0.05; **p < 0.01; ****p < 0.0001. Data are presented as mean and error bars represent SD.

Figure 7.

A schematic overview of the impact of C. sporogenes on the course of intestinal inflammation.