Bindloss et al nitrobenzanilide(Gw9662)mimicked the reduction(P<0.05)in Angptl4 transcript levels observed after streptomycin treatment(Fig. IG). The effects of each treatment on epithel Nos2 expression( Fig. 1H) were opposite to those observed for expression of Angptl4 (Fig IG), which supported the idea that PPAR-y negatively regulates Nos2(Fig. SI) Next, we used E. coli indicator strains to investigate whether silencing PPAR-y signaling would increase the bioavailability of nitrate in the colon. To this end, mice were inoculated with a 1: I mixture of a nitrate respiration-proficient indicator strain(E. coli wild type)and an isogenic nitrate respiration-deficient indicator strain(napa narG narZ mutant). Treatment ith the PPAR-y agonist rosiglitazone abrogated the fitness advantage conferred to wild- (Fig. IA) expansion of E. coli without antibiotic treatment, mice were mock-treated (inoculation with sterile PBS)or treated with the PPAR-y antagonist Gw9662 and then infected with E. coll indicator strains. Treatment with Gw9662 significantly increased the overall number of E. coli recovered from the colon of mice(P<0.05)(Fig. ID) by driving a nitrate respiration dependent E coli expansion, as shown by increased recovery of the wild type over a nitrate espiration-deficient mutant(P<0.05)(Fig. IJ). Next, we wanted to determine whether treatment with a PPAr-y antagonist would increase the abundance of endogenous Enterobacteriaceae. While endogenous Enterobacteriaceae were not detected in C57BL/6 mice from Jackson, C57BL/6 mice from Charles River carried endogenous E coli strains producing nitrate reductase activity(Fig. S4A). Treatment of Charles river mice with iw9662 significantly(P<0.05)increased the abundance of endogenous E. coli, which could be abrogated by supplementation with the iNOS inhibitor AG(Fig. $4B) Epithelial PPAR-y-signaling limits luminal nitrate availability To exclude the possibility that our results were due to off-target effects of chemical agonist or antagonists, we generated mice lacking PPAR-y in the intestinal epithelium (Pparg/i villifrel- mice)along wild-type littermate control animals(Ppargfitl villin /- mice). Mice lacking epithelial PPAR-y signaling exhibited significantly elevated transcript levels of Nos2 in the colonic epithelium(P<0.01)(Fig. 2A), which resulted neither from reduced abundance of butyrate-producing bacteria in their gut microbiota(Fig. 2B and Fig s5)nor from lower butyrate levels in their cecal contents(Fig. 2C). Inoculation with E. coll indicator strains revealed that epithelial PPAR-y-deficiency increased the bioavailability of nitrate through a mechanism that required iNOS activity, because treatment with the iNOS inhibitor AG abrogated the nitrate respiration-dependent growth advantage(P<0.05)(Fig 2D). Similar results were obtained when mice were infected with the murine E. coli isolate 32 (Fig. S4C), which produced nitrate reductase activity(Fig. S4A). To test directly hether genetic ablation of epithelial PPAR-y-signaling increased the concentration of nitrate in the intestinal lumen, we measured the concentration of this electron acceptor colonic mucus scrapings, which revealed a significant increase(P<0.01) in mice lacking epithelial PPAR-y signaling compared to littermate controls( Fig. 2E) Mice lacking epithelial PPAR-y-signaling were treated with streptomycin, infected the next day with E. coli indicator strains and inoculated one day later with a community of 17 Science Author manuscript; available in PMC 2017 October 1nitrobenzanilide (GW9662) mimicked the reduction (P < 0.05) in Angptl4 transcript levels observed after streptomycin treatment (Fig. 1G). The effects of each treatment on epithelial Nos2 expression (Fig. 1H) were opposite to those observed for expression of Angptl4 (Fig. 1G), which supported the idea that PPAR-γ negatively regulates Nos2 (Fig. S1). Next, we used E. coli indicator strains to investigate whether silencing PPAR-γ signaling would increase the bioavailability of nitrate in the colon. To this end, mice were inoculated with a 1:1 mixture of a nitrate respiration-proficient indicator strain (E. coli wild type) and an isogenic nitrate respiration-deficient indicator strain (napA narG narZ mutant). Treatment with the PPAR-γ agonist rosiglitazone abrogated the fitness advantage conferred to wild￾type E. coli by nitrate respiration in streptomycin-treated mice (Fig. 1A). To investigate whether inhibition of PPAR-γ signaling would support a nitrate respiration-dependent expansion of E. coli without antibiotic treatment, mice were mock-treated (inoculation with sterile PBS) or treated with the PPAR-γ antagonist GW9662 and then infected with E. coli indicator strains. Treatment with GW9662 significantly increased the overall number of E. coli recovered from the colon of mice (P < 0.05) (Fig. 1I) by driving a nitrate respiration￾dependent E. coli expansion, as shown by increased recovery of the wild type over a nitrate respiration-deficient mutant (P < 0.05) (Fig. 1J). Next, we wanted to determine whether treatment with a PPAR-γ antagonist would increase the abundance of endogenous Enterobacteriaceae. While endogenous Enterobacteriaceae were not detected in C57BL/6 mice from Jackson, C57BL/6 mice from Charles River carried endogenous E. coli strains producing nitrate reductase activity (Fig. S4A). Treatment of Charles River mice with GW9662 significantly (P < 0.05) increased the abundance of endogenous E. coli, which could be abrogated by supplementation with the iNOS inhibitor AG (Fig. S4B). Epithelial PPAR-γ-signaling limits luminal nitrate availability To exclude the possibility that our results were due to off-target effects of chemical agonist or antagonists, we generated mice lacking PPAR-γ in the intestinal epithelium (Pparg fl/flVillin cre/− mice) along wild-type littermate control animals (Pparg fl/flVillin −/− mice). Mice lacking epithelial PPAR-γ signaling exhibited significantly elevated transcript levels of Nos2 in the colonic epithelium (P < 0.01) (Fig. 2A), which resulted neither from a reduced abundance of butyrate-producing bacteria in their gut microbiota (Fig. 2B and Fig. S5) nor from lower butyrate levels in their cecal contents (Fig. 2C). Inoculation with E. coli indicator strains revealed that epithelial PPAR-γ-deficiency increased the bioavailability of nitrate through a mechanism that required iNOS activity, because treatment with the iNOS inhibitor AG abrogated the nitrate respiration-dependent growth advantage (P < 0.05) (Fig. 2D). Similar results were obtained when mice were infected with the murine E. coli isolate JB2 (Fig. S4C), which produced nitrate reductase activity (Fig. S4A). To test directly whether genetic ablation of epithelial PPAR-γ-signaling increased the concentration of nitrate in the intestinal lumen, we measured the concentration of this electron acceptor in colonic mucus scrapings, which revealed a significant increase (P < 0.01) in mice lacking epithelial PPAR-γ signaling compared to littermate controls (Fig. 2E). Mice lacking epithelial PPAR-γ-signaling were treated with streptomycin, infected the next day with E. coli indicator strains and inoculated one day later with a community of 17 Byndloss et al. Page 3 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript