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 13). 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