Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion

Abstract

Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator-activated receptor γ (PPAR-γ). Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-γ signaling. Microbiota-induced PPAR-γ signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward β-oxidation. Therefore, microbiota-activated PPAR-γ signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.

Figure 1. The PPAR-γ agonist butyrate limits the availability of nitrate by repressing Nos2 expression

(A) Streptomycin (Strep)-treated mice (N = 8) 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 tissue culture medium received IFNγ/IL-22, Rosi or AG treatment. Nitrate produced in the apical compartment was determined by a modified Griess assay. (C–F) Mice (N = 8) were mock-treated (inoculation with vehicle control) or treated with Strep and organs collected 3 days later. (C) The abundance of Clostridia in colon contents was determined by quantitative real-time PCR using class-specific primers for Clostridia 16S rRNA genes. (D) Relative abundance of families belonging to the class Clostridia determined by 16S profiling of DNA isolated from colon contents. (E) The butyrate concentration was determined in cecal contents using gas chromatography and (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) 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–I) Bars represent geometric means ± standard error. (A, B and J) Dots represent measurements from individual animals (A and J) or wells (B) and bars represent geometric means. *, P < 0.05^{; **,} P < 0.01.

Figure 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 Pparg^fl/flVillin^cre/− mice (Pparg), which lack PPAR-γ in epithelial cells, and in littermate control Pparg^fl/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) The butyrate concentration was determined in cecal contents of mice (N = 6) using 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; the ratio of wt and napA narG narZ mutant recovered from colon contents) was determined 3 days after inoculation. (E) The 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 ± standard error. (D, E and G) Each dot represents data from an individual animal and black bars represent geometric means. *, P < 0.05^{; **,} P < 0.01; ns, not statistically significantly different.

Figure 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 isolated one 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 one 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 counter stained with DAPI nuclear stain (blue fluorescence). (G) Representative images are shown. Scale bars represent 50 μm. (H) A veterinary pathologist scored blinded sections for hypoxia staining. Each dot represents data from one animal. (A-B) Bars represent geometric means ± standard error. (C–F) Each dot 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.

Figure 4. Microbiota-induced PPAR-γ-signaling and T_regs 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 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 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–G) Groups of mice (N = 6) were treated with anti-CD25 antibody or isotype control antibody and colonocytes isolated to measure intracellular concentrations of lactate (E), ATP (F) or mitochondrial cytochrome c oxidase activity. (G) Boxes in Whisker plots represent the standard error, while lines indicate the standard deviation. (A–B and E–F) Bars represent geometric means ± standard error. (C, D and H) Each dot 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.