Gut commensals-derived succinate impels colonic inflammation in ulcerative colitis

Abstract

Gut microbiota-derived metabolites play a crucial role in modulating the inflammatory response in inflammatory bowel disease (IBD). In this study, we identify gut microbiota-derived succinate as a driver of inflammation in ulcerative colitis (UC) by activating succinate-responsive, colitogenic helper T (Th) cells that secrete interleukin (IL)-9. We demonstrate that colitis is associated with an increase in succinate-producing gut bacteria and decrease in succinate-metabolizing gut bacteria. Similarly, UC patients exhibit elevated levels of succinate-producing gut bacteria and luminal succinate. Intestinal colonization by succinate-producing gut bacteria or increased succinate availability, exacerbates colonic inflammation by activating colitogenic Th9 cells. In contrast, intestinal colonization by succinate-metabolizing gut bacteria, blocking succinate receptor signaling with an antagonist, or neutralizing IL-9 with an anti-IL-9 antibody alleviates inflammation by reducing colitogenic Th9 cells. Our findings underscore the role of gut microbiota-derived succinate in driving colitogenic Th9 cells and suggesting its potential as a therapeutic target for treating IBD.

Fig. 1. Colitis mice and UC patients displayed elevated succinate levels. Treatment with a succinate receptor (SUCNR1) inhibitor in mice led to a decrease in inflammation.

A Volcano plots showing differential stool metabolites comparing colitis with healthy controls. B Volcano plots showing differential serum metabolites comparing colitis with healthy controls. C, D Volcano plots showing differential stool and serum metabolites comparing UC with healthy volunteers. E The hallmark metabolite, succinate, was significantly upregulated in both the stool and serum compartments of colitis mice and UC patients. The numbers represent various metabolites, with only succinate consistently elevated across all samples. F FACS data showing an increased frequency of SUCNR1⁺ IL-9⁺ CD4⁺ T cells in the lamina propria of Oxazolone-colitis mice compared to healthy mice. G Schematic illustrating the treatment regimen for SUCNR1 inhibition in oxazolone-induced colitis mice, using compound 7a. H Body weight recovery in oxazolone-induced colitis mice treated with compound 7a surpassed that of untreated mice. I Inflammatory lesion formation was significantly suppressed in oxazolone-induced colitis mice treated with compound 7a compared to untreated mice. J The expression levels of Th9-associated genes, including IL-9, Foxo1, IL-9R, and BATF, were quantified using qPCR from the MLNs of mice treated with compound 7a compared to untreated mice. K The expression level of SUCNR1, a surrogate indicator for succinate availability, was measured using qPCR in the MLNs of mice treated with compound 7a compared to untreated mice. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (Student’s t-test or one-way ANOVA).

Fig. 2. The colon tissue-resident microbiota triggers succinate-mediated pro-inflammatory responses in mice with oxazolone-induced colitis.

A Schematic of mouse gut microbiome depletion using an antibiotic cocktail [amphotericin-B (0.1 mg/ml; Sigma), ampicillin (1 g/l; Sigma), vancomycin (5 mg/ml; Sigma), metronidazole (10 mg/ml; Sigma), and neomycin (10 mg/ml; Sigma)]. B Body weight reduction is less pronounced in AIMD mice than in wild-type mice following colitis induction with oxazolone. C Inflammatory lesion formation is significantly suppressed in AIMD mice compared to wild-type mice after colitis induction. D Representative hematoxylin and eosin-stained sections of mouse colon tissue, accompanied by blinded histologic scores (scale bar: 100 μm). E Fecal succinate levels measured in various groups of animals. F Flow cytometry plots of CD4⁺IL-9⁺ helper T cells from the colonic lamina propria, along with percentages. G The expression levels of Th9-associated genes, including IL-9, Foxo1, IL-9R, and BATF, were quantified using qPCR from the MLNs of treated mice. H The expression level of SUCNR1, a surrogate indicator of succinate availability, was measured using qPCR in the MLNs of treated mice. I Schematic of fecal succinate elevation using FMT from diseased and streptomycin donors (a concentration of 20 mg in 200 μl of PBS by gavage per mouse for 72 h, followed by colitis induction using oxazolone). J Succinate levels measured in stools from diseased and streptomycin-donor mice. K Body weight reduction in FMT donors. L Inflammatory lesion formation in FMT donors. M Body weight reduction in FMT recipients. N Inflammatory lesion formation in stool recipients from oxazolone donors and oxazolone-streptomycin-donors. O Representative hematoxylin and eosin-stained sections of mouse colon tissue from recipients of oxazolone and oxazolone-streptomycin stools, accompanied by blinded histologic scores. P Succinate levels were measured in the stools of recipients of oxazolone and oxazolone-streptomycin stools. Q The expression levels of Th9-associated genes, including IL-9, Foxo1, IL-9R, BATF, AHR, and IRF-4, were quantified using qPCR from the MLNs of recipients of oxazolone and oxazolone-streptomycin stools. R The expression level of SUCNR1, a surrogate indicator of succinate availability, was measured using qPCR in the MLNs of recipients of oxazolone and oxazolone-streptomycin stools. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (Student’s t-test, one-way ANOVA, or two-way ANOVA).

Fig. 3. Ulcerative colitis disrupts the balance of gut microbes involved in succinate production and metabolism in both humans and mice.

A Schematic of stool specimen collection for metagenomics and qPCR. B Heatmap showing the top ten microbiota families with their respective relative abundances (n = 4). Bacteroidaceae was abundant in the Oxa-colitis group compared to the other families. C Venn diagram showing the relative abundance (%) of top families between healthy controls and Oxa-colitis. D Heat tree analysis depicting alterations in microbiota composition between healthy controls and Oxa-colitis. Significantly altered taxa are displayed by name at the corresponding node. Nodes indicate the hierarchical structure of taxa. A blue branch indicates a decrease in healthy controls compared to Oxa-colitis. E Relative abundance of individual species of the Bacteroidaceae family in stool specimens collected from healthy controls and the Oxa-colitis group. F Relative abundance of individual species of the Acidaminococcaceae family in stool specimens collected from healthy controls and the Oxa-colitis group. G Compared to healthy individuals, UC patients have a significant increase in the abundance of Bacteroidaceae, the major producers of succinate in the gut microbiota, and a decrease in Acidaminococcaceae, which is important for succinate metabolism. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (One-way ANOVA or two-way ANOVA).

Fig. 4. Colonization of Bacteroides vulgatus increases IL-9-secreting CD4⁺ T cells and exacerbates oxazolone-colitis due to an elevation in fecal succinate.

A Schematic of Bacteroides vulgatus colonization and colitis induction. B Fecal succinate measurement using LC–MS in healthy controls, Oxa alone, and Oxa-colonized with Bacteroides vulgatus. C Body weight recovery in healthy controls, Oxa alone, and Oxa-colonized with Bacteroides vulgatus (n = 6). D Formation of colonic lesions in healthy controls, Oxa alone, and Oxa-colonized with Bacteroides vulgatus (n = 6). E Representative images of distal colons stained with H&E and respective statistics for pathology scores. F Representative FACS plot for IL-9 measurement in helper T cells harvested from the mesentery of healthy controls, oxazolone-treated mice, and Oxa-colonized with Bacteroides vulgatus (n = 6). G Representative qPCR data for the expression of IL-9 and related genes Foxo1, PU.1, and IL-9R. H The expression level of SUCNR1, a surrogate indicator for succinate availability, was measured using qPCR in the MLNs of healthy controls, Oxa alone, and Oxa-colonized with Bacteroides vulgatus. I Schematic showing colonization of Bacteroides vulgatus followed by anti-IL-9 and SUCNR1 antagonist (7a) treatment. J Reduction in body weight in healthy controls, Oxa alone, Oxa-colonized with Bacteroides vulgatus, Oxa-colonized with Bacteroides vulgatus plus 7a, and Oxa-colonized with Bacteroides vulgatus plus anti-IL-9 treatment (n = 6). K Formation of colonic lesions in healthy controls, Oxa alone, Oxa-colonized with Bacteroides vulgatus, Oxa-colonized with Bacteroides vulgatus plus 7a, and Oxa-colonized with Bacteroides vulgatus plus anti-IL-9 treatment (n = 6). L Representative images of distal colons stained with H&E and respective statistics for pathology scores of various groups. M Representative qPCR data for the expression of IL-9 and associated genes in healthy controls, Oxa alone, Oxa-colonized with Bacteroides vulgatus, Oxa-colonized with Bacteroides vulgatus plus 7a, and Oxa-colonized with Bacteroides vulgatus plus anti-IL-9 treatment (n = 6). N The expression level of SUCNR1, a surrogate indicator for succinate availability, was measured using qPCR in the MLNs of healthy controls, Oxa alone, Oxa-colonized with Bacteroides vulgatus, Oxa-colonized with Bacteroides vulgatus plus 7a, and Oxa-colonized with Bacteroides vulgatus plus anti-IL-9 treatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (One-way ANOVA or two-way ANOVA).

Fig. 5. Supplementing with retinoic acid disrupts the colonization of Bacteroides vulgatus, thereby ameliorating Oxa-colitis through the reduction of fecal succinate levels.

A Representative qPCR data depicting the relative abundance of Bacteroides vulgatus in stool samples from mice with and without retinoic acid (RA) supplementation. B Schematic diagram showing the regimen for RA treatment and colitis induction. C The reduction in body weight in healthy controls, Oxa alone, and Oxa supplemented with RA. D Formation of colonic lesions in healthy controls, Oxa alone, and Oxa supplemented with retinoic acid. E Representative images of distal colons stained with H&E, along with respective statistics for pathology scores. F Representative FACS plot for IL-9 measurement in helper T cells harvested from the mesentery of healthy controls, Oxa alone, and Oxa supplemented with RA (n = 6). G Representative qPCR data for the expression of IL-9 and associated genes in healthy controls, Oxa alone, and Oxa supplemented with RA. H PCA plot showing distinct metabolic profiles in stool samples collected from healthy controls, Oxa alone, and Oxa supplemented with RA. I Hierarchical clustering showing distinct clusters that effectively differentiate the metabolomic profiles of stool samples from healthy, Oxa-colitis, and RA-treated Oxa-colitis mice. J The heatmap illustrates stool metabolite analysis from healthy controls, Oxa alone, and Oxa supplemented with RA. Supplemented animals showed a significant reduction in fecal succinate levels. K Correlation of identified metabolites with each other. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (Student’s t-test or one-way ANOVA or two-way ANOVA).

Fig. 6. The succinate consuming bacterial consortium and individual species protect against oxazolone-induced colitis.

A Schematic showing the colonization of the consortium and individual species enriched in succinate consumers. B Reduction in body weight of healthy control, Oxa alone, and Oxa-colonized with Phascolarctobacterium succinatutens (n = 6). C Reduction in body weight of healthy control, Oxa alone, and Oxa-colonized with Dialister succinatiphilus (n = 6). D Reduction in body weight of healthy control, Oxa alone, and Oxa-colonized with consortia (n = 6). E Colonic lesions in Oxa alone, Oxa-colonized with Phascolarctobacterium succinatutens, Oxa-colonized with Dialister succinatiphilus, and Oxa-colonized with consortia (n = 6). F Fecal succinate was measured in individual strains and consortia-treated groups of animals. G Representative images of distal colons stained with H&E and respective statistics for pathology scores. H Representative FACS plot for IL-9 measurement in helper T cells harvested from the mesentery of healthy control, oxazolone, Oxa-colonized with Phascolarctobacterium succinatutens, Oxa-colonized with Dialister succinatiphilus, and Oxa-colonized with consortia. I–K Representative qPCR data for the expression of IL-9 and associated genes for Th9 and succinate (SUCNR1) in healthy control, Oxa alone, Oxa-colonized with Phascolarctobacterium succinatutens, Oxa-colonized with Dialister succinatiphilus, and Oxa-colonized with consortia. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA or two-way ANOVA).