HHS Public access Author manuscript Science. Author manuscript; available in PMC 2017 October 16 Published in final edited form as Science. 2017 August 11; 357(6351): 570-575. doi: 10. 1 126/science. aam9949 Microbiota-activated PPAR-y-signaling inhibits dysbiotic Enterobacteriaceae expansion Mariana X. Byndloss', Erin E. Olsan', Fabian Rivera-Chavezl, Connor R. Tiffany Stephanie A Cevallos, Kristen L Lokken, Teresa P. Torres, Austin J. ByndlossT Franziska Faber Yandong Gao2, Yael Litvak', Christopher A Lopez, Gege Xu3, Eleonora Napoli, Cecilia Giulivi, Renee M. Solis, Alexander Revzin, Carlito Lebrilla,and Andreas j Baumler 1, Department of Medical Microbiology and Immunology, School of Medicine, University of California at davis One shields Ave: Davis ca 95616. USA 2Department of Biomedical Engineering, College of Engineering, University of California at Davis One Shields Ave: Davis CA 95616 USA Department of Chemistry, College of Letters and Sciences, University of California at Davis, One Shields Ave: Davis CA 95616 USA Department of Molecular Biosciences, School of Veterinary Medicine, University of California at Davis. One Shields Ave: Davis CA 95616. USA Abstract Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found depletion of butyrate- producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor PPAR-y. Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase(iNOS)was elevated in the absence of PPAR-y-signaling. Microbiota-induced PPAR-y-signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes)towards p-oxidation. Therefore, microbiota-activated PPAR-y-signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon a balanced gut microbiota is characterized by the dominance of obligate anaerobic members of the phyla Firmicutes and Bacteroidetes, while an expansion of facultative anaerobic Enterobacteriaceae(phylum Proteobacteria)is a common marker of gut dysbiosis(1)(Fig S1). Obligate anaerobic bacteria prevent dysbiotic expansion of facultative anaerobic Enterobacteriaceae, in part, by limiting the generation of host-derived nitrate and oxygen(2 To whom correspondence should be addressed. ajbaumler( @ucdavis. edu. Materials and Methods Figs. SI to S6
Microbiota-activated PPAR-γ-signaling inhibits dysbiotic Enterobacteriaceae expansion Mariana X. Byndloss1, Erin E. Olsan1, Fabian Rivera-Chávez1, Connor R. Tiffany1, Stephanie A. Cevallos1, Kristen L. Lokken1, Teresa P. Torres1, Austin J. Byndloss1, Franziska Faber1, Yandong Gao2, Yael Litvak1, Christopher A. Lopez1, Gege Xu3, Eleonora Napoli4, Cecilia Giulivi4, Renée M. Tsolis1, Alexander Revzin2, Carlito Lebrilla3, and Andreas J. Bäumler1,* 1Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Ave; Davis CA 95616, USA 2Department of Biomedical Engineering, College of Engineering, University of California at Davis, One Shields Ave; Davis CA 95616, USA 3Department of Chemistry, College of Letters and Sciences, University of California at Davis, One Shields Ave; Davis CA 95616, USA 4Department of Molecular Biosciences, School of Veterinary Medicine, University of California at Davis, One Shields Ave; Davis CA 95616, USA Abstract Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found depletion of butyrateproducing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor PPAR-γ. Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase (iNOS) was elevated in the absence of PPAR-γ-signaling. Microbiota-induced PPAR-γ-signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) towards β-oxidation. Therefore, microbiota-activated PPAR-γ-signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon. A balanced gut microbiota is characterized by the dominance of obligate anaerobic members of the phyla Firmicutes and Bacteroidetes, while an expansion of facultative anaerobic Enterobacteriaceae (phylum Proteobacteria) is a common marker of gut dysbiosis (1) (Fig. S1). Obligate anaerobic bacteria prevent dysbiotic expansion of facultative anaerobic Enterobacteriaceae, in part, by limiting the generation of host-derived nitrate and oxygen (2, *To whom correspondence should be addressed. ajbaumler@ucdavis.edu. Supplemental Materials: Materials and Methods Figs. S1 to S6 HHS Public Access Author manuscript Science. Author manuscript; available in PMC 2017 October 16. Published in final edited form as: Science. 2017 August 11; 357(6351): 570–575. doi:10.1126/science.aam9949. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Bindloss et al 3). It is not known which host-signaling pathways are triggered by the gut microbiota to limit the availability of these respiratory electron acceptors. We found disruption of the gut microbiota by streptomycin treatment increased the bioavailability of host-derived nitrate the lumen of the large intestine. Increased recovery of a wild-type E. coli strain by comparison with an isogenic derivative deficient for nitrate respiration(napA narG narz mutant)was observed in mice(C57BL/6 from Jackson) infected with a 1: 1 mixture of both strains(Fig. 1A). Supplementing streptomycin-treated mice with the iNOS inhibitor aminoguanidine hydrochloride(Ag)abrogated the growth advantage conferred upon E. coll by nitrate respiration(Fig. IA), supporting the notion that luminal nitrate was host-derived To model nitrate production by the colonic epithelium, we induced NOS2 expression in human colonic epithelial cancer( Caco2)cells by stimulation with gamma interferon(IFNy) and interleukin (IL)-22(model epithelia). We exposed the model epithelia to butyrate, a fermentation product of the gut microbiota that serves as the main carbon source of colonic epithelial cells(colonocytes)(5). Butyrate significantly reduced NOS2 expression(P<0.05) (Fig. S2A), lowered INOS synthesis(P<0.05)(6)(Fig. S2B)and diminished epithelial generation of nitrate(P<0.05)(Fig. 1B), a product of nitric oxide decomposition in the intestinal lumen(4). The host can sense butyrate using the nuclear receptor PPAR-Y, whi is synthesized at high levels in colonocytes ()and does not respond to other short-chain fatty acids, such as acetate or propionate(8). To determine whether PPAR-y repressed INOS synthesis, we stimulated model epithelia with the PPAR-y agonist rosiglitazone Rosiglitazone-treatment significantly blunted NOS2 expression(P<0.05)(Fig. S2A), reduced INOS synthesis(P<0.05)(Fig. S2B), lowered nitrate production(P<0.01)(Fig I B)and induced synthesis and nuclear localization of PPAR-y in model epithelia(Fig S2C). Based on these data we hypothesized that microbiota-derived butyrate suppresses lOs synthesis in the gut by stimulating PPAR-y-signaling in colonocytes(Fig. S1) Streptomycin-mediated depletion of a PPAR-y agonist drives growth by nitrate respiration To test our hypothesis, we used mice to investigate whether streptomycin treatment would deplete butyrate-producing bacteria, thereby increasing Nos2 expression in colonocytes Streptomycin treatment reduced bacterial numbers in colon contents(Fig. S3A)and significantly(P<0.01)reduced the abundance of Clostridia(phylum Firmicutes)(Fig. IC and $3B), which are obligate anaerobes that include abundant butyrate-producers(9)(Fig S3C), specifically Lachnospiraceae and Ruminococcaceae(Fig. ID and S3D). The changes in the microbiota composition correlated with a significant(P<0.01)drop in the cecal butyrate concentration(Fig. 1E)and significantly(P<0.05)elevated Nos2 expression in murine colonocyte preparations( Fig. IF) We next investigated the role of PPAR-y in altering epithelial gene expression. Streptomycin treatment reduced epithelial expression of Angptl4, a gene positively regulated by PPAR-Y (8), and expression was restored in streptomycin-treated mice that received the PPAR-y agonist rosiglitazone(Fig. IG). Treatment of mice with the PPAR-y antagonist 2-chloro-5- Science Author manuscript; available in PMC 2017 October 1
3). It is not known which host-signaling pathways are triggered by the gut microbiota to limit the availability of these respiratory electron acceptors. We found disruption of the gut microbiota by streptomycin treatment increased the bioavailability of host-derived nitrate in the lumen of the large intestine. Increased recovery of a wild-type E. coli strain by comparison with an isogenic derivative deficient for nitrate respiration (napA narG narZ mutant) was observed in mice (C57BL/6 from Jackson) infected with a 1:1 mixture of both strains (Fig. 1A). Supplementing streptomycin-treated mice with the iNOS inhibitor aminoguanidine hydrochloride (AG) abrogated the growth advantage conferred upon E. coli by nitrate respiration (Fig. 1A), supporting the notion that luminal nitrate was host-derived (2, 4). To model nitrate production by the colonic epithelium, we induced NOS2 expression in human colonic epithelial cancer (Caco2) cells by stimulation with gamma interferon (IFNγ) and interleukin (IL)-22 (model epithelia). We exposed the model epithelia to butyrate, a fermentation product of the gut microbiota that serves as the main carbon source of colonic epithelial cells (colonocytes) (5). Butyrate significantly reduced NOS2 expression (P < 0.05) (Fig. S2A), lowered iNOS synthesis (P < 0.05)(6) (Fig. S2B) and diminished epithelial generation of nitrate (P < 0.05) (Fig. 1B), a product of nitric oxide decomposition in the intestinal lumen (4). The host can sense butyrate using the nuclear receptor PPAR-γ, which is synthesized at high levels in colonocytes (7) and does not respond to other short-chain fatty acids, such as acetate or propionate (8). To determine whether PPAR-γ repressed iNOS synthesis, we stimulated model epithelia with the PPAR-γ agonist rosiglitazone. Rosiglitazone-treatment significantly blunted NOS2 expression (P < 0.05) (Fig. S2A), reduced iNOS synthesis (P < 0.05) (Fig. S2B), lowered nitrate production (P < 0.01) (Fig. 1B) and induced synthesis and nuclear localization of PPAR-γ in model epithelia (Fig. S2C). Based on these data we hypothesized that microbiota-derived butyrate suppresses iNOS synthesis in the gut by stimulating PPAR-γ-signaling in colonocytes (Fig. S1). Streptomycin-mediated depletion of a PPAR-γ agonist drives growth by nitrate respiration To test our hypothesis, we used mice to investigate whether streptomycin treatment would deplete butyrate-producing bacteria, thereby increasing Nos2 expression in colonocytes. Streptomycin treatment reduced bacterial numbers in colon contents (Fig. S3A) and significantly (P < 0.01) reduced the abundance of Clostridia (phylum Firmicutes) (Fig. 1C and S3B), which are obligate anaerobes that include abundant butyrate-producers (9) (Fig. S3C), specifically Lachnospiraceae and Ruminococcaceae (Fig. 1D and S3D). The changes in the microbiota composition correlated with a significant (P < 0.01) drop in the cecal butyrate concentration (Fig. 1E) and significantly (P < 0.05) elevated Nos2 expression in murine colonocyte preparations (Fig. 1F). We next investigated the role of PPAR-γ in altering epithelial gene expression. Streptomycin treatment reduced epithelial expression of Angptl4, a gene positively regulated by PPAR-γ (8), and expression was restored in streptomycin-treated mice that received the PPAR-γ agonist rosiglitazone (Fig. 1G). Treatment of mice with the PPAR-γ antagonist 2-chloro-5- Byndloss et al. Page 2 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
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 1
nitrobenzanilide (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 wildtype 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 respirationdependent 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
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 1
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. 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 microbiotaderived 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
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 1
PPAR-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 DSStreated 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 microbiotaderived 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