Figure 3.
Consortia of succinate-producing bacteria increase colonic TCs and type 2 cytokines and corresponds to higher colonic concentrations of succinate. (A–E) IL-25, IL-13, and IL-5 protein concentrations were measured by ELISA (see Materials and methods) in the colon (D and E). IL-25 protein concentrations were also measured in the cecum and the ileum of C57BL/6 WT mice receiving succinate-producing or non-succinate-producing bacterial consortia. Protein concentration in the lysate (pg/ml) was normalized by total protein in the sample (in mg) as in Buonuomo et al. (2016). Data are representative of independent experiments repeated twice, using n = 4 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine significant differences. Statistical significance was estimated at an FDR of 0.05. (F) TC hyperplasia was estimated via flow cytometry in the colon and ileum of AVNM-treated C57BL/6 WT mice that were administered a consortium of three succinate producers, three succinate non-producers, or PBS. TC ratios were assessed by gating on CD45-Epcam+Siglecf+ cells. Data are representative of independent experiments repeated twice, using n = 4–5 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine significant differences. Statistical significance was estimated at FDR of 0.05. (G) TC hyperplasia in the colon was confirmed via immunohistochemistry by enumerating the number of DCLK1-expressing cells in the field of view (FOV). The white bar indicates a scale of 100 μm. Data are representative of independent experiments repeated twice, using n = 4 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine significant differences. Statistical significance was estimated at FDR of 0.05. (H) To demonstrate that phenotype induction corresponded to colonization by the bacterial consortia, we performed shotgun metagenomic sequencing. The relative abundances of the top 10 abundant species plus others are displayed as stacked bar plots and indicate colonic engraftment by the administered treatment consortia in the respective recipient mice. As expected, almost no colonization is observed in the ileum. (I) Targeted metabolomics was performed to quantify concentrations of acetic acid, propionic acid, butyric acid, succinic acid, 2-methylbutryic acid, isolaveric acid, valeric acid, and succinic acid in the colon and the ileum of AVNM-treated C57BL/6 WT mice receiving the succinate- or non-succinate-producing bacterial consortia. Data are representative of independent experiments repeated twice, using n = 4 female mice per treatment group. Two-sample t test was run to determine differentially abundant metabolites. Statistical significance was estimated at an FDR of 0.05. Note: Animals assigned to treatment with different consortia were first cohoused to homogenize the microbiome before AVNM treatment and then separated according to the type of bacterial consortium administered.

Consortia of succinate-producing bacteria increase colonic TCs and type 2 cytokines and corresponds to higher colonic concentrations of succinate. (A–E) IL-25, IL-13, and IL-5 protein concentrations were measured by ELISA (see Materials and methods) in the colon (D and E). IL-25 protein concentrations were also measured in the cecum and the ileum of C57BL/6 WT mice receiving succinate-producing or non-succinate-producing bacterial consortia. Protein concentration in the lysate (pg/ml) was normalized by total protein in the sample (in mg) as in Buonuomo et al. (2016). Data are representative of independent experiments repeated twice, using n = 4 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine significant differences. Statistical significance was estimated at an FDR of 0.05. (F) TC hyperplasia was estimated via flow cytometry in the colon and ileum of AVNM-treated C57BL/6 WT mice that were administered a consortium of three succinate producers, three succinate non-producers, or PBS. TC ratios were assessed by gating on CD45-Epcam+Siglecf+ cells. Data are representative of independent experiments repeated twice, using n = 4–5 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine significant differences. Statistical significance was estimated at FDR of 0.05. (G) TC hyperplasia in the colon was confirmed via immunohistochemistry by enumerating the number of DCLK1-expressing cells in the field of view (FOV). The white bar indicates a scale of 100 μm. Data are representative of independent experiments repeated twice, using n = 4 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine significant differences. Statistical significance was estimated at FDR of 0.05. (H) To demonstrate that phenotype induction corresponded to colonization by the bacterial consortia, we performed shotgun metagenomic sequencing. The relative abundances of the top 10 abundant species plus others are displayed as stacked bar plots and indicate colonic engraftment by the administered treatment consortia in the respective recipient mice. As expected, almost no colonization is observed in the ileum. (I) Targeted metabolomics was performed to quantify concentrations of acetic acid, propionic acid, butyric acid, succinic acid, 2-methylbutryic acid, isolaveric acid, valeric acid, and succinic acid in the colon and the ileum of AVNM-treated C57BL/6 WT mice receiving the succinate- or non-succinate-producing bacterial consortia. Data are representative of independent experiments repeated twice, using n = 4 female mice per treatment group. Two-sample t test was run to determine differentially abundant metabolites. Statistical significance was estimated at an FDR of 0.05. Note: Animals assigned to treatment with different consortia were first cohoused to homogenize the microbiome before AVNM treatment and then separated according to the type of bacterial consortium administered.

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