Figure 4.
Colonic induction of TCs and related cytokines is dependent on the presence of succinate-producing B. thetaiotaomicron and Pou2f3-dependent tuft cells. AVNM-treated mice were orally gavaged with heat-killed B. thetaiotaomicron, live WT B. thetaiotaomicron, succinate production-deficient B. thetaiotaomicron Δfrd, or PBS. (A) TC hyperplasia in the colon of the mice receiving each treatment was assessed via IHC 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. (B) TC percentage in the colon and the ileum of AVNM-treated mice gavaged with heat-killed B. thetaiotaomicron, live B. thetaiotaomicron, or B. thetaiotaomicron Δfrd was evaluated via flow cytometry by gating on CD45−Epcam+Siglecf+ cells. 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. (C) Relative mRNA expression measured by RT-qPCR of IL-25 in the proximal colon of AVNM-treated C57BL/6 WT mice treated with live B. thetaiotaomicron or B. thetaiotaomicron Δfrd. 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. (D) Shotgun metagenomic sequencing was performed from colonic and ileal samples to evaluate colonization by B. thetaiotaomicron and succinate production-deficient B. thetaiotaomicron Δfrd. We confirmed the engraftment of B. thetaiotaomicron Δfrd by mapping metagenomic reads to the B. thetaiotaomicron frd gene using bowtie2 and counting how many reads were mapped to frd in each treatment. (E) Targeted metabolomics to estimate 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 administered heat-killed B. thetaiotaomicron, B. thetaiotaomicron WT, or succinate production-deficient B. thetaiotaomicron Δfrd. 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 metabolites significantly different between treatments. Statistical significance was estimated at an FDR of 0.05. (F and G) Type 1, type 3, and G type 2–associated cytokines measured by Luminex Multiplex ELISA in the proximal colon of AVNM-treated C57BL/6 WT mice treated with B. thetaiotaomicron or B. thetaiotaomicron Δfrd. Data are representative of experiments repeated twice, using n = 4–8 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine metabolites significantly different between treatments. Statistical significance was estimated at FDR of 0.05. (H) Estimation of TC percentage the colon and ileum of AVNM-treated Pou2f3+/−, Pou2f3−/−, and Pou2f3+/+ mice treated with B. thetaiotaomicron or B. thetaiotaomicron Δfrd. TC percentage was estimated via flow cytometry by gating on CD45−Epcam+Siglecf+ cells. We run ANOVA comparing the linear model with interaction, TC ∼ Genotype + Treatment + Genotype:Treatment against the model with no interaction TC ∼ Genotype + Treatment, to evaluate Pou2f3-dependent hyperplasia of TCs in response to different microbial treatments. Pou2f3+/− and Pou2f3−/− were littermates. Data are representatives of experiments repeated twice, using n = 4 female mice per treatment group. Pou2f3+/−, Pou2f3−/− were littermate, while Pou2f3+/+ from a different litter. Pou2f3+/−, Pou2f3−/−, and Pou2f3+/+ mice were co-housed for at least 2 wk for basal microbiome equilibration before antibiotic treatment and administration of bacteria. (I) Relative mRNA expression measured by RT-qPCR of IL-25 in the colon and ileum of AVNM-pretreated Pou2f3−/− and Pou2f3+/+ mice treated with B. thetaiotaomicron or B. thetaiotaomicron Δfrd. Same modeling approach as in H was applied. Data are representatives of experiments repeated twice, using n = 4 female mice per treatment group. Pou2f3−/− and Pou2f3+/+ mice were co-housed for at least 2 wk for basal microbiome equilibration before antibiotic treatment and administration of bacteria.

Colonic induction of TCs and related cytokines is dependent on the presence of succinate-producing B . thetaiotaomicron and Pou2f3-dependent tuft cells. AVNM-treated mice were orally gavaged with heat-killed B. thetaiotaomicron, live WT B. thetaiotaomicron, succinate production-deficient B. thetaiotaomicron Δfrd, or PBS. (A) TC hyperplasia in the colon of the mice receiving each treatment was assessed via IHC 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. (B) TC percentage in the colon and the ileum of AVNM-treated mice gavaged with heat-killed B. thetaiotaomicron, live B. thetaiotaomicron, or B. thetaiotaomicron Δfrd was evaluated via flow cytometry by gating on CD45−Epcam+Siglecf+ cells. 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. (C) Relative mRNA expression measured by RT-qPCR of IL-25 in the proximal colon of AVNM-treated C57BL/6 WT mice treated with live B. thetaiotaomicron or B. thetaiotaomicron Δfrd. 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. (D) Shotgun metagenomic sequencing was performed from colonic and ileal samples to evaluate colonization by B. thetaiotaomicron and succinate production-deficient B. thetaiotaomicron Δfrd. We confirmed the engraftment of B. thetaiotaomicron Δfrd by mapping metagenomic reads to the B. thetaiotaomicron frd gene using bowtie2 and counting how many reads were mapped to frd in each treatment. (E) Targeted metabolomics to estimate 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 administered heat-killed B. thetaiotaomicron, B. thetaiotaomicron WT, or succinate production-deficient B. thetaiotaomicron Δfrd. 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 metabolites significantly different between treatments. Statistical significance was estimated at an FDR of 0.05. (F and G) Type 1, type 3, and G type 2–associated cytokines measured by Luminex Multiplex ELISA in the proximal colon of AVNM-treated C57BL/6 WT mice treated with B. thetaiotaomicron or B. thetaiotaomicron Δfrd. Data are representative of experiments repeated twice, using n = 4–8 female mice per treatment group. ANOVA with Tukey post-hoc test was run to determine metabolites significantly different between treatments. Statistical significance was estimated at FDR of 0.05. (H) Estimation of TC percentage the colon and ileum of AVNM-treated Pou2f3+/−, Pou2f3−/−, and Pou2f3+/+ mice treated with B. thetaiotaomicron or B. thetaiotaomicron Δfrd. TC percentage was estimated via flow cytometry by gating on CD45−Epcam+Siglecf+ cells. We run ANOVA comparing the linear model with interaction, TC ∼ Genotype + Treatment + Genotype:Treatment against the model with no interaction TC ∼ Genotype + Treatment, to evaluate Pou2f3-dependent hyperplasia of TCs in response to different microbial treatments. Pou2f3+/− and Pou2f3−/− were littermates. Data are representatives of experiments repeated twice, using n = 4 female mice per treatment group. Pou2f3+/−, Pou2f3−/− were littermate, while Pou2f3+/+ from a different litter. Pou2f3+/−, Pou2f3−/−, and Pou2f3+/+ mice were co-housed for at least 2 wk for basal microbiome equilibration before antibiotic treatment and administration of bacteria. (I) Relative mRNA expression measured by RT-qPCR of IL-25 in the colon and ileum of AVNM-pretreated Pou2f3−/− and Pou2f3+/+ mice treated with B. thetaiotaomicron or B. thetaiotaomicron Δfrd. Same modeling approach as in H was applied. Data are representatives of experiments repeated twice, using n = 4 female mice per treatment group. Pou2f3−/− and Pou2f3+/+ mice were co-housed for at least 2 wk for basal microbiome equilibration before antibiotic treatment and administration of bacteria.

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