Bindloss et al PPAR-y-signaling activates mitochondrial p-oxidation in alternatively activated(M2 macrophages(13). Since IFNy-signaling drives the energy metabolism of macrophages towards anaerobic glycolysis(13), we hypothesized that in addition to silencing PPAR-y signaling, metabolic reprogramming of colonocytes might also require an inflammator signal(Fig. S1). To test this idea, we turned to S. enterica serovar Typhimurium(S Typhimurium), a pathogen that employs two type Ill secretion systems to trigger intestinal inflammation and uses the cyxAB genes, encoding cytochrome bc-ll oxidase, for bsequent aerobic expansion in the intestinal lumen(3). When mice were infected with S Typhimurium strains that were proficient(wild type)or deficient(cyxA mutant) for aerobic respiration under microaerophilic conditions, a benefit provided by aerobic respiration was observed in mice lacking epithelial PPAR-y-signaling, but not in littermate control animals (Fig. 3E). To investigate whether inflammatory responses elicited by Salmonella virulence factors were required to increase the bioavailability of oxygen, we inactivated the two type II secretion systems essential for Salmonella enteropathogenicity through mutations in InvA and spiB (3). Consistent with our hypothesis, aerobic respiration no longer provided a benefit to the pathogen in mice lacking epithelial PPAR-y-signaling when mice were infected with avirulent S. Typhimurium strains that were either proficient(invA spiB mutant)or deficient(invA spiB cyx A mutant) for aerobic respiration under microaerophilic conditions( Fig. 3E) To test whether, in addition to genetic ablation of PPAR-y-signaling, an inflammatory signal was needed to increase luminal oxygen bioavailability, mice received low-dose(1%in drinking water)dextran sodium sulfate(DSs)-treatment, which elicited inflammatory hanges as indicated by a reduction in colon length(Fig. S6C). Inoculation with E. coll indicator strains revealed that aerobic respiration provided a larger growth benefit in DSS- treated mice lacking epithelial PPAR-y-signaling compared to their Dss-treated littermate controls(Fig. 3D). Similarly, inoculation with avirulent S.Typhimurium strains that were either proficient(inv A spiB mutant)or deficient(inv A spiB cyxA mutant) for aerobic respiration under microaerophilic conditions provided evidence for increased oxygen bioavailability only in DSS-treated mice that lacked epithelial PPAR-y-signaling(Fig 3F) Next, we investigated whether either Dss treatment or infection with wild-type S Typhimurium would increase epithelial oxygenation in mice lacking epithelial PPAR-Y ignaling. To this end, we visualized the hypoxia of sur hypoxic marker pimonidazole, which is reduced under hypoxic conditions to hydroxylamine intermediates that irreversibly bind to nucleophilic groups in proteins or DNA (14, 15) Genetic ablation of PPAR-y-signaling was not sufficient to reduce epithelial hypoxia. However, DSS-treatment or infection with wild-type S. Typhimurium increased epithelial oxygenation in mice lacking epithelial PPAR-y-signaling, while hypoxia staining remained unchanged in littermate control animals(Fig. 3G and 3H) PPAR-y-signaling and T regs cooperate to maintain colonocyte hy poxia While streptomycin treatment reduced PPAR-y-signaling(Fig. IG) by depleting microbiot derived butyrate(Fig. IE and S6B), the findings shown above suggested that reducing PPAR-y-signaling was necessary, but not sufficient for increasing oxygen bioavailability in Science Author manuscript; available in PMC 2017 October 1PPAR-γ-signaling activates mitochondrial β-oxidation in alternatively activated (M2) macrophages (13). Since IFNγ-signaling drives the energy metabolism of macrophages towards anaerobic glycolysis (13), we hypothesized that in addition to silencing PPAR-γ- signaling, metabolic reprogramming of colonocytes might also require an inflammatory signal (Fig. S1). To test this idea, we turned to S. enterica serovar Typhimurium (S. Typhimurium), a pathogen that employs two type III secretion systems to trigger intestinal inflammation and uses the cyxAB genes, encoding cytochrome bd-II oxidase, for its subsequent aerobic expansion in the intestinal lumen (3). When mice were infected with S. Typhimurium strains that were proficient (wild type) or deficient (cyxA mutant) for aerobic respiration under microaerophilic conditions, a benefit provided by aerobic respiration was observed in mice lacking epithelial PPAR-γ-signaling, but not in littermate control animals (Fig. 3E). To investigate whether inflammatory responses elicited by Salmonella virulence factors were required to increase the bioavailability of oxygen, we inactivated the two type III secretion systems essential for Salmonella enteropathogenicity through mutations in invA and spiB (3). Consistent with our hypothesis, aerobic respiration no longer provided a benefit to the pathogen in mice lacking epithelial PPAR-γ-signaling when mice were infected with avirulent S. Typhimurium strains that were either proficient (invA spiB mutant) or deficient (invA spiB cyxA mutant) for aerobic respiration under microaerophilic conditions (Fig. 3E). To test whether, in addition to genetic ablation of PPAR-γ-signaling, an inflammatory signal was needed to increase luminal oxygen bioavailability, mice received low-dose (1% in drinking water) dextran sodium sulfate (DSS)-treatment, which elicited inflammatory changes as indicated by a reduction in colon length (Fig. S6C). Inoculation with E. coli indicator strains revealed that aerobic respiration provided a larger growth benefit in DSS￾treated mice lacking epithelial PPAR-γ-signaling compared to their DSS-treated littermate controls (Fig. 3D). Similarly, inoculation with avirulent S.. Typhimurium strains that were either proficient (invA spiB mutant) or deficient (invA spiB cyxA mutant) for aerobic respiration under microaerophilic conditions provided evidence for increased oxygen bioavailability only in DSS-treated mice that lacked epithelial PPAR-γ-signaling (Fig. 3F). Next, we investigated whether either DSS treatment or infection with wild-type S. Typhimurium would increase epithelial oxygenation in mice lacking epithelial PPAR-γ- signaling. To this end, we visualized the hypoxia of surface colonocytes using the exogenous hypoxic marker pimonidazole, which is reduced under hypoxic conditions to hydroxylamine intermediates that irreversibly bind to nucleophilic groups in proteins or DNA (14, 15). Genetic ablation of PPAR-γ-signaling was not sufficient to reduce epithelial hypoxia. However, DSS-treatment or infection with wild-type S. Typhimurium increased epithelial oxygenation in mice lacking epithelial PPAR-γ-signaling, while hypoxia staining remained unchanged in littermate control animals (Fig. 3G and 3H). PPAR-γ-signaling and Tregs cooperate to maintain colonocyte hypoxia While streptomycin treatment reduced PPAR-γ-signaling (Fig. 1G) by depleting microbiota￾derived butyrate (Fig. 1E and S6B), the findings shown above suggested that reducing PPAR-γ-signaling was necessary, but not sufficient for increasing oxygen bioavailability in Byndloss et al. Page 5 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript