the colon, because DSS-induced inflammation or Salmonella virulence factors were required for increasing colonocyte oxygenation(Fig. 3D-3H). Although streptomycin treatment does not lead to overt inflammation, disruption of the gut microbiota reduced concentrations of other microbiota-derived short-chain fatty acids( Fig. S6D) besides butyrate( Fig. S6B), that signal through G-protein-coupled receptor(GPR)43, GPR109A and histone deacetylases expressed by T cells, dendritic cells and macrophages, respectively, to reduce intestinal inflammation(16-19). Engagement of these host cell receptors by short-chain fatty acids induces maturation and expansion of regulatory T-cells (Tregs) in the colon, a cell type that limits pro-inflammatory responses(20-24). Consistent with previous reports showing that antibiotic-mediated depletion of short-chain fatty acids leads to a contraction of the Treg population in the colonic mucosa(20, 23), we observed that streptomycin treatment shran the pool of colonic Tregs(CD3*-enriched CD4* FOXP3t cells)to one third of its normal size (Fig. 4A, S6E and S7). Thus, during antibiotic treatment, the second input that increases oxygen bioavailability in the colon might be provided by contraction of the colonic Treg population, which increases the inflammatory tone of the mucosa(Fig. Sl) Treatment with anti-CD25 antibody reduced the pool of colonic Tregs(Fig 4B and S6F)by a magnitude similar to that observed after streptomycin treatment(Fig. 4A)and elicited inflammatory changes in mice lacking epithelial PPAR-y-signaling, as indicated by a reduction in colon length(Fig. S6G). When anti-CD25-treated 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, there was no benefit provided by aerobic respiration to S. Typhimurium in wild-type mice. Hence, depletion of Tregs was not sufficient for increasing oxygen bioavailability. In contrast depletion of Tregs increased oxygen bioavailability in mice lacking epithelial PPAR-y- signaling, but not in wild-type littermate control mice(Fig. 4C and S6H). Genetic ablation of PPAR-y-signaling combined with Treg-depletion phenocopied the effects of streptomycin treatment on the recovery of avirulent S. Typhimurium strains proficient or deficient for aerobic respiration under microaerophilic conditions(Fig. 4D). Depletion of Tregs increased epithelial oxygenation in mice lacking epithelial PPAR-y-signaling, but not in littermate control mice(Fig. 3G and 3H). Consistent with metabolic reprogramming towards anaerobic glycolysis, Treg-depletion increased intracellular lactate levels and lowered ATP concentrations in colonocyte preparations from mice lacking epithelial PPAR-y-signaling, but not from littermate controls( Fig. 4E and 4F). Measurement of mitochondrial cytochrome c oxidase activity revealed that Treg-depletion caused a significant (P<0.01) eduction in oxygen consumption in colonocyte preparations of mice lacking epithelial PPAR-y-signaling, but not in littermate control animals(Fig. 4G) To further study how colonic Tregs and PPAR-y-signaling cooperate to limit respiratory growth of facultative anaerobic bacteria, mice lacking epithelial PPAR-y-signaling were mutant lacking cytochrome bd oxidase and nitrate reductases(cydAB napA narG narl 9 treated with anti-CD25 antibody and infected with a 1: I mixture of wild-type E coli and mutant). The competitive index was approximately 1,000-fold greater(P<0.o1)in anti CD25-treated mice lacking epithelial PPAR-y compared to wild-type littermate control animals(Fig. 4H). Similar results were obtained when mice were infected with individual bacterial strains(Fig. S6D) Science Author manuscript; available in PMC 2017 October 1the colon, because DSS-induced inflammation or Salmonella virulence factors were required for increasing colonocyte oxygenation (Fig. 3D–3H). Although streptomycin treatment does not lead to overt inflammation, disruption of the gut microbiota reduced concentrations of other microbiota-derived short-chain fatty acids (Fig. S6D) besides butyrate (Fig. S6B), that signal through G-protein-coupled receptor (GPR)43, GPR109A and histone deacetylases expressed by T cells, dendritic cells and macrophages, respectively, to reduce intestinal inflammation (16–19). Engagement of these host cell receptors by short-chain fatty acids induces maturation and expansion of regulatory T-cells (Tregs) in the colon, a cell type that limits pro-inflammatory responses (20–24). Consistent with previous reports showing that an antibiotic-mediated depletion of short-chain fatty acids leads to a contraction of the Treg population in the colonic mucosa (20, 23), we observed that streptomycin treatment shrank the pool of colonic Tregs (CD3+-enriched CD4+ FOXP3+ cells) to one third of its normal size (Fig. 4A, S6E and S7). Thus, during antibiotic treatment, the second input that increases oxygen bioavailability in the colon might be provided by contraction of the colonic Treg population, which increases the inflammatory tone of the mucosa (Fig. S1). Treatment with anti-CD25 antibody reduced the pool of colonic Tregs (Fig. 4B and S6F) by a magnitude similar to that observed after streptomycin treatment (Fig. 4A) and elicited inflammatory changes in mice lacking epithelial PPAR-γ-signaling, as indicated by a reduction in colon length (Fig. S6G). When anti-CD25-treated 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, there was no benefit provided by aerobic respiration to S. Typhimurium in wild-type mice. Hence, depletion of Tregs was not sufficient for increasing oxygen bioavailability. In contrast, depletion of Tregs increased oxygen bioavailability in mice lacking epithelial PPAR-γ- signaling, but not in wild-type littermate control mice (Fig. 4C and S6H). Genetic ablation of PPAR-γ-signaling combined with Treg-depletion phenocopied the effects of streptomycin treatment on the recovery of avirulent S. Typhimurium strains proficient or deficient for aerobic respiration under microaerophilic conditions (Fig. 4D). Depletion of Tregs increased epithelial oxygenation in mice lacking epithelial PPAR-γ-signaling, but not in littermate control mice (Fig. 3G and 3H). Consistent with metabolic reprogramming towards anaerobic glycolysis, Treg-depletion increased intracellular lactate levels and lowered ATP concentrations in colonocyte preparations from mice lacking epithelial PPAR-γ-signaling, but not from littermate controls (Fig. 4E and 4F). Measurement of mitochondrial cytochrome c oxidase activity revealed that Treg-depletion caused a significant (P < 0.01) reduction in oxygen consumption in colonocyte preparations of mice lacking epithelial PPAR-γ-signaling, but not in littermate control animals (Fig. 4G). To further study how colonic Tregs and PPAR-γ-signaling cooperate to limit respiratory growth of facultative anaerobic bacteria, mice lacking epithelial PPAR-γ-signaling were treated with anti-CD25 antibody and infected with a 1:1 mixture of wild-type E. coli and a mutant lacking cytochrome bd oxidase and nitrate reductases (cydAB napA narG narZ mutant). The competitive index was approximately 1,000-fold greater (P < 0.01) in anti￾CD25-treated mice lacking epithelial PPAR-γ compared to wild-type littermate control animals (Fig. 4H). Similar results were obtained when mice were infected with individual bacterial strains (Fig. S6I). Byndloss et al. Page 6 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript