human Clostridia isolates (10). Inoculation with the Clostridia isolates restored cecal butyrate concentrations(P<0.01)(Fig. 2F)and suppressed nitrate respiration-dependent growth of E. coll in streptomycin-treated littermate control mice. However, nitrate respiration-dependent growth of E. coli was not suppressed in streptomycin-treated mice lacking epithelial PPAR-y-signaling(P<0.05)(Fig. 2G). To directly test whether butyrate were treated with streptomycin, infected the next day with E. coli indicator strains and inoculated one day later with 1, 2, 3-tributyrylglycerol (tributyrin). Tributyrin, a natural ingredient of butter, exhibits delayed absorption in the small intestine compared to butyrate and its degradation in the large intestine increases luminal butyrate concentrations (11) Tributyrin supplementation restored cecal butyrate concentrations(P<0.01)(Fig. 2F), which abrogated nitrate respiration-dependent growth of E. coli in streptomycin-treated littermate control mice, but not in streptomycin-treated mice lacking epithelial PPAR-y signaling(P<0.05)(Fig. 2G). Collectively, these data support the idea that microbiota- derived butyrate maintains gut homeostasis by inducing epithelial PPAR-y-signaling, which in turn limits nitrate respiration-dependent dysbiotic E. coli expansion(Fig. S1) Lack of epithelial PPAR-y-signaling increases colonocyte oxygenation during colitis Colonocytes obtain energy through B-oxidation of microbiota-derived butyrate, which consumes a considerable amount of oxygen, thereby rendering the epithelium hypoxic(12) However, after a streptomycin-mediated depletion of butyrate(ig. IE), colonocytes switch their energy metabolism to converting glucose into lactate(anaerobic glycolysis)(I1) Consistent with this metabolic reprogramming, streptomycin treatment increased the concentration of lactate(Fig 3A)and reduced ATP levels(Fig. 3B)in primary murine colonocyte preparations. Anaerobic glycolysis does not consume oxygen, which then permeates through the epithelium into the gut lumen(3, 11). This scenario was supported by increased recovery of aerobic respiration-proficient(Nissle 1917 wild type)E. coli over E. coli strain that is impaired for aerobic respiration under microaerophilic conditions (cydAB mutant) from the colon of streptomycin-treated mice(Fig. 3C). Similar results were obtained when the result was repeated with a different E. coli strain(MG1655)(Fig. S6A) Increasing the concentration of the PPAR-y antagonist butyrate in streptomycin-treated mice either by inoculation with a community of 17 human Clostridia isolates or by supplementation with tributyrin(Fig. S6B)appeared to reduce the bioavailability of oxygen, as E. coli indicator strains that were proficient(wild type)or deficient(cydAB mutant)for aerobic respiration under microaerophilic conditions were recovered equally in the gut(Fig 3C). Furthermore, the aerobic growth benefit observed in streptomycin-treated mice inoculated with E coli indicator strains was abrogated by treatment with the Ppar\,the agonist rosiglitazone(Fig 3C). Surprisingly, E. coli indicator strains were recovered at ratio from mice lacking epithelial PPAR-y signaling and from their littermate controls (Fig. 3D), suggesting that reducing PPAR-y signaling alone was not sufficient for increasing he bioavailability of oxygen Science Author manuscript; available in PMC 2017 October 1human Clostridia isolates (10). Inoculation with the Clostridia isolates restored cecal butyrate concentrations (P < 0.01) (Fig. 2F) and suppressed nitrate respiration-dependent growth of E. coli in streptomycin-treated littermate control mice. However, nitrate respiration-dependent growth of E. coli was not suppressed in streptomycin-treated mice lacking epithelial PPAR-γ-signaling (P < 0.05) (Fig. 2G). To directly test whether butyrate was responsible for inhibiting nitrate respiration of E. coli in littermate control animals, mice were treated with streptomycin, infected the next day with E. coli indicator strains and inoculated one day later with 1,2,3-tributyrylglycerol (tributyrin). Tributyrin, a natural ingredient of butter, exhibits delayed absorption in the small intestine compared to butyrate and its degradation in the large intestine increases luminal butyrate concentrations (11). Tributyrin supplementation restored cecal butyrate concentrations (P < 0.01) (Fig. 2F), which abrogated nitrate respiration-dependent growth of E. coli in streptomycin-treated littermate control mice, but not in streptomycin-treated mice lacking epithelial PPAR-γ- signaling (P < 0.05) (Fig. 2G). Collectively, these data support the idea that microbiota￾derived butyrate maintains gut homeostasis by inducing epithelial PPAR-γ-signaling, which in turn limits nitrate respiration-dependent dysbiotic E. coli expansion (Fig. S1). Lack of epithelial PPAR-γ-signaling increases colonocyte oxygenation during colitis Colonocytes obtain energy through β-oxidation of microbiota-derived butyrate, which consumes a considerable amount of oxygen, thereby rendering the epithelium hypoxic (12). However, after a streptomycin-mediated depletion of butyrate (Fig. 1E), colonocytes switch their energy metabolism to converting glucose into lactate (anaerobic glycolysis) (11). Consistent with this metabolic reprogramming, streptomycin treatment increased the concentration of lactate (Fig. 3A) and reduced ATP levels (Fig. 3B) in primary murine colonocyte preparations. Anaerobic glycolysis does not consume oxygen, which then permeates through the epithelium into the gut lumen (3, 11). This scenario was supported by increased recovery of aerobic respiration-proficient (Nissle 1917 wild type) E. coli over an E. coli strain that is impaired for aerobic respiration under microaerophilic conditions (cydAB mutant) from the colon of streptomycin-treated mice (Fig. 3C). Similar results were obtained when the result was repeated with a different E. coli strain (MG1655) (Fig. S6A). Increasing the concentration of the PPAR-γ antagonist butyrate in streptomycin-treated mice either by inoculation with a community of 17 human Clostridia isolates or by supplementation with tributyrin (Fig. S6B) appeared to reduce the bioavailability of oxygen, as E. coli indicator strains that were proficient (wild type) or deficient (cydAB mutant) for aerobic respiration under microaerophilic conditions were recovered equally in the gut (Fig. 3C). Furthermore, the aerobic growth benefit observed in streptomycin-treated mice inoculated with E. coli indicator strains was abrogated by treatment with the PPAR-γ agonist rosiglitazone (Fig. 3C). Surprisingly, E. coli indicator strains were recovered at the same ratio from mice lacking epithelial PPAR-γ signaling and from their littermate controls (Fig. 3D), suggesting that reducing PPAR-γ signaling alone was not sufficient for increasing the bioavailability of oxygen. Byndloss et al. Page 4 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript