Research Article

Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion

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Science  11 Aug 2017:
Vol. 357, Issue 6351, pp. 570-575
DOI: 10.1126/science.aam9949
  • Fig. 1 The PPAR-γ agonist butyrate limits the availability of nitrate by repressing Nos2 expression.

    (A) Streptomycin (Strep)–treated mice (N = 8 animals) were inoculated with a 1:1 mixture of E. coli wild type (wt) and napA narG narZ mutant and received rosiglitazone (Rosi) or aminoguanidine (AG) supplementation. The competitive index [(CI), the ratio of wt and napA narG narZ mutant recovered from colon contents] was determined 3 days after inoculation. (B) Polarized Caco-2 cells (N = 4) grown in a tissue culture medium received IFN-γ/IL-22, Rosi, or AG treatment. Nitrate produced in the apical compartment was determined by a modified Griess assay. (C to F) Mice (N = 8) were mock-treated (inoculated with vehicle control) or treated with Strep, and organs were collected 3 days later. (C) Abundance of Clostridia in colon contents was determined by quantitative real-time polymerase chain reaction (PCR) using class-specific primers for Clostridia 16S ribosomal RNA genes. (D) Relative abundance of families belonging to the class Clostridia determined by 16S profiling of DNA isolated from colon contents. (E) Butyrate concentration was determined in cecal contents by gas chromatography. (F) Transcript level of Nos2 in colonocyte preparations was determined by real-time PCR. (G and H) Colonocytes were isolated 3 days after treatment with streptomycin from mice (N = 8) receiving the indicated supplementation and transcript levels of Angptl4 (G) and Nos2 (H), as determined by quantitative real-time PCR. (I and J) Mice (N = 6) were mock-treated or received the PPAR-γ antagonist GW9662 and were inoculated with E. coli indicator strains. Numbers of E. coli (I) and the CI of indicator strains (J) were determined 3 days after inoculation. (C and E to I) Bars represent geometric means ± SE. (A, B, and J) Circles represent measurements from individual animals [(A) and (J)] or wells (B), and bars represent geometric means. *P < 0.05; **P < 0.01.

  • Fig. 2 Microbiota-induced epithelial PPAR-γ signaling limits nitrate availability in the colon.

    (A) Nos2 expression in the colonic epithelium of mice (N = 6) was determined by real-time PCR in Ppargfl/flVillincre/– mice (Pparg), which lack PPAR-γ in epithelial cells, and in littermate control Ppargfl/flVillin/ mice (WT). (B) Relative abundance of families belonging to the class Clostridia in colon contents of mice (N = 6) was determined by 16S profiling. (C) Butyrate concentration was determined in cecal contents of mice (N = 6) by gas chromatography. (D) Mice (N = 6) were inoculated with a 1:1 mixture of E. coli wild type (wt) and napA narG narZ mutant and received aminoguanidine (AG) supplementation or vehicle control. The competitive index (CI) was determined 3 days after inoculation. (E) Concentration of nitrate in the colonic mucus layer was determined in groups of animals (N = 9) by a modified Griess assay. (F and G) Streptomycin-treated mice (N = 6) were inoculated with E. coli indicator strains and received supplementation with tributyrin or a community of 17 human Clostridia isolates (C17). The butyrate concentration in cecal contents (F) and the CI in colon contents (G) were determined 3 days after inoculation. (A, C, and F) Bars represent geometric means ± SE. (D, E, and G) Each circle represents data from an individual animal, and black bars represent geometric means. *P < 0.05; **P < 0.01; ns, not statistically significantly different.

  • Fig. 3 Lack of epithelial PPAR-γ signaling increases colonocyte oxygenation during colitis.

    (A and B) Groups of mice (N = 5) were mock-treated or treated with streptomycin (Strep), and colonocytes were isolated 1 day later to measure intracellular concentrations of lactate (A) or ATP (B). (C) Groups (N = 6) of streptomycin-treated or mock-treated mice were inoculated with a 1:1 mixture of E. coli wild type (wt) and cydAB mutant and received supplementation with rosiglitazone (Rosi), tributyrin, or a community of 17 human Clostridia isolates (C17). (D) Groups of mice (N = 6) receiving no supplementation or water supplemented with 1% dextran sulfate sodium (DSS) were inoculated with a 1:1 mixture of E. coli wild type (wt) and cydAB mutant. (C and D) The competitive index (CI) was determined 3 days after inoculation. (E and F) Groups of mice (N = 6) were inoculated with the indicated Salmonella strain mixtures. (F) Mice received no supplementation or water supplemented with 1% DSS. (G and H) Mice (N = 6) were treated as indicated and were injected intraperitoneally with pimonidazole 1 hour before euthanasia. Binding of pimonidazole was detected using hypoxyprobe-1 primary antibody and a Cy-3 conjugated goat anti-mouse secondary antibody (red fluorescence) in sections of the colon that were counterstained with DAPI (4′,6-diamidino-2-phenylindole) nuclear stain (blue fluorescence). (G) Representative images. Scale bars, 50 μm. (H) A veterinary pathologist scored blinded sections for hypoxia staining. Each circle represents data from one animal. (A and B) Bars represent geometric means ± SE. (C to F) Each circle represents data from an individual animal, and black bars represent geometric means. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not statistically significantly different.

  • Fig. 4 Microbiota-induced PPAR-γ signaling and Tregs cooperate to limit the bioavailability of oxygen in the colon.

    (A and B) Groups of mice (N = 4) were treated with streptomycin (A) or with anti-CD25 antibody (B), and CD3+-enriched live colonic cells were analyzed for expression of CD4 and FOXP3 by flow cytometry. (C) Groups of mice (N = 6) were treated with anti-CD25 antibody or isotype control and, 10 days later, were inoculated with a 1:1 mixture of an avirulent Salmonella strain (invA spiB mutant) and an avirulent Salmonella strain lacking cytochrome bdII oxidase (invA spiB cyxA mutant). (D) Groups (N = 6) of streptomycin-treated or mock-treated mice were inoculated with Salmonella indicator strains and received supplementation with tributyrin or a community of 17 human Clostridia isolates (C17). (C and D). The CI was determined 4 days after inoculation. (E to G) Groups of mice (N = 6) were treated with anti-CD25 antibody or isotype control antibody, and colonocytes were isolated to measure intracellular concentrations of lactate (E), ATP (F), or mitochondrial cytochrome c oxidase activity (G). (G) The width of the box shows the interquartile range, the horizontal line shows the median, and the top and bottom lines show the highest and lowest values, respectively. (H) Groups (N = 6) of anti-CD25–treated mice lacking epithelial PPAR-γ signaling or untreated wild-type mice (WT) were infected with a 1:1 mixture of the indicated E. coli strains. The CI was determined 4 days after inoculation. (A, B, E, and F) Bars represent geometric means ± SE. (C, D, and H) Each circle represents data from an individual animal, and black bars represent geometric means. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not statistically significantly different.

Supplementary Materials

  • Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion

    Mariana X. Byndloss, Erin E. Olsan, Fabian Rivera-Chávez, Connor R. Tiffany, Stephanie A. Cevallos, Kristen Lokken, Teresa P. Torres, Austin J. Byndloss, Franziska Faber, Yandong Gao, Yael Litvak, Christopher A. Lopez, Gege Xu, Eleonora Napoli, Cecilia Giulivi, Renée M. Tsolis, Alexander Revzin, Carlito B. Lebrilla, Andreas J. Bäumler

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S7
    • References

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