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 1
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 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 antiCD25-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
Bindloss et al the microbial community towards a dominance of obligate anaerobes, which produce short chain fatty acids. In turn, short-chain fatty acids sustain Tregs and epithelial PPAR-y signaling, which cooperatively drives the energy metabolism of colonocytes towards B- oxidation of microbiota-derived butyrate to preserve epithelial hypoxia, thereby closing a virtuous cycle maintaining homeostasis of a healthy gut. PPAR-y-signaling also activates expression of beta-defensins, which might contribute to shaping the intestinal environment (25). An antibiotic-induced lack of epithelial PPAR-y-signaling and a contraction of the treg pool cooperatively drive a metabolic reorientation of colonocytes towards anaerobic glycolysis, thereby increasing epithelial oxygenation and consequently elevating oxygen bioavailability to promote an expansion of Enterobacteriaceae(Fig. SD), a common marker of dysbiosis(1). Thus, an expansion of Enterobacteriaceae in the gut-associated microbial community is a microbial signature of epithelial dysfunction, which has important ramifications for targeting PPAR-y-signaling as a potential intervention strategy MATERIALS AND METHODS Bacterial culture conditions The 17 human Clostridia isolates were kindly provided by K. Honda(20, 26)and were cultured individually as described previously (10). E. coli strains( wild-type E coli Nissle 1917, E. coli Nissle 1917 napA narZ narG mutant (2), E. coli Nissle 1917 cydAB mutant, E. coll Nissle 1917 cydAB napA narZ narG mutant, E colI JB2 (27), E colI MG1655, E. coll MG1655 cydA mutant)and S. Typhmurium strains(wild-type ATCC14028 resistant to nalidixic acid []. Ir715 invA spiB mutant, IR715 cyxA mutant or IR715 InvA spiB cyxA mutant)(3)were routinely grown aerobically at 37 C in LB broth(BD Biosciences)or on LB plates. When necessary, antibiotics were added to the media at the following concentrations: 0. I mg/ml carbenicillin, and 0.05 mg/ml kanamycin Single colonies of endogenous coliforms isolated on McConkey agar from the feces of Charles river mice were subjected to species identification using EnteroPluri Test (Liofilchem, Italy ) per the manufacturer's recommendations Construction of e. coli mutants To construct an E coli Nissle 1917 cydA mutant, upstream and downstream regions of approximately 0.5kb in length flanking the cyda gene were amplified by PCr and purified using the MiniElute kit( Qiagen). The pRDH1O suicide vector was digested with Sall, run on an agarose gel, purified using the MiniElute kit(Qiagen) and assembled with the fragments using the Gibson Assembly Master Mix(NEB)to form plasmid pYl9 Plasmid pYL9 was nd conjugation performed with E. coli Nissle 1917 carrying the temperature-sensitive plasmid pSw172 for counter selection(4). Conjugation was performed at 30oC and exconjugants in which the suicide plasmid had integrated into the chromosome were recovered on LB plates containing carbenicilin and chloramphenicol Subsequent sucrose selection was performed on sucrose plates(5% sucrose,& g/l nutrient broth base, 15 g/l agar)to select for a second crossover events. PCR was performed to detect events that lead to the unmarked deletion of the cydA gene Plasmid pswI72 was cured by Science Author manuscript; available in PMC 2017 October 1
The emerging picture is that epithelial hypoxia maintains anaerobiosis in the colon to drive the microbial community towards a dominance of obligate anaerobes, which produce shortchain fatty acids. In turn, short-chain fatty acids sustain Tregs and epithelial PPAR-γ- signaling, which cooperatively drives the energy metabolism of colonocytes towards β- oxidation of microbiota-derived butyrate to preserve epithelial hypoxia, thereby closing a virtuous cycle maintaining homeostasis of a healthy gut. PPAR-γ-signaling also activates expression of beta-defensins, which might contribute to shaping the intestinal environment (25). An antibiotic-induced lack of epithelial PPAR-γ-signaling and a contraction of the Treg pool cooperatively drive a metabolic reorientation of colonocytes towards anaerobic glycolysis, thereby increasing epithelial oxygenation and consequently elevating oxygen bioavailability to promote an expansion of Enterobacteriaceae (Fig. S1), a common marker of dysbiosis (1). Thus, an expansion of Enterobacteriaceae in the gut-associated microbial community is a microbial signature of epithelial dysfunction, which has important ramifications for targeting PPAR-γ-signaling as a potential intervention strategy. MATERIALS AND METHODS Bacterial culture conditions The 17 human Clostridia isolates were kindly provided by K. Honda (20, 26) and were cultured individually as described previously (10). E. coli strains (wild-type E. coli Nissle 1917, E. coli Nissle 1917 napA narZ narG mutant (2), E. coli Nissle 1917 cydAB mutant, E. coli Nissle 1917 cydAB napA narZ narG mutant, E. coli JB2 (27), E. coli MG1655, E. coli MG1655 cydA mutant) and S. Typhmurium strains (wild-type ATCC14028 resistant to nalidixic acid [IR715], IR715 invA spiB mutant, IR715 cyxA mutant or IR715 invA spiB cyxA mutant) (3) were routinely grown aerobically at 37°C in LB broth (BD Biosciences) or on LB plates. When necessary, antibiotics were added to the media at the following concentrations: 0.1 mg/ml carbenicillin, and 0.05 mg/ml kanamycin. Single colonies of endogenous coliforms isolated on McConkey agar from the feces of Charles River mice were subjected to species identification using EnteroPluri Test (Liofilchem, Italy) per the manufacturer`s recommendations. Construction of E. coli mutants To construct an E. coli Nissle 1917 cydA mutant, upstream and downstream regions of approximately 0.5kb in length flanking the cydA gene were amplified by PCR and purified using the MiniElute kit (Qiagen). The pRDH10 suicide vector was digested with SalI, run on an agarose gel, purified using the MiniElute kit (Qiagen) and assembled with the fragments using the Gibson Assembly Master Mix (NEB) to form plasmid pYL9. Plasmid pYL9 was then transformed into E. coli S17-1 λpir and conjugation performed with E. coli Nissle 1917 carrying the temperature-sensitive plasmid pSW172 for counter selection (4). Conjugation was performed at 30°C and exconjugants in which the suicide plasmid had integrated into the chromosome were recovered on LB plates containing carbenicilin and chloramphenicol. Subsequent sucrose selection was performed on sucrose plates (5 % sucrose, 8 g/l nutrient broth base, 15 g/l agar) to select for a second crossover events. PCR was performed to detect events that lead to the unmarked deletion of the cydA gene. Plasmid pSW172 was cured by Byndloss et al. Page 7 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript