Bindloss et al For Treg depletion, mice were intraperitoneally injected with 250 ug/mouse of PC61 mAb or isotype control, as previously described (28). At day 10 after treatment, mice were inoculated with 1 x 10%CFU of a 1: I mixture of the indicated E coli or S. Typhimurium strains. Samples were collected at 4 days after infection For microbiota analysis, mice were euthanized at 3 days after streptomycin treatment, and DNA from the colon contents was extracted using the Power Soil DNA Isolation kit (Mo Bio)according to the manufacturers protocol. Generation and analysis of sequencing data is described below Colonocyte isolation The proximal colon was flushed with ice-cold phosphate buffered saline(PBs)(Gibco using a 10 mL syringe fitted with an 18-gauge blunt needle before opening lengthwise, cutting into 2- cm pieces and placing in a 15 mL conical centrifuge tube filled with 10 mL ce-cold PBS(Gibco) on ice. Tubes were gently inverted four times, PBS(Gibco) wash was removed and replaced with additional ice-cold PBS(Gibco) before transfer to sterile petri dishes where colon tissue was cut into <5 mm pieces using a razor blade. Tissue pieces were placed in crypt chelating buffer(2 mM EDTA, pH 8 in PBS)and incubated for 30 minutes with gentle rocking buried in ice. Chelating buffer was removed and replaced with cold dissociation buffer (54.9 mM D-sorbitol, 43. 4 mM sucrose in PBS) before shaking vigorously by hand for 8 minutes. Cell suspension was transferred to a new 15 mL conical centrifuge tube, leaving any large remnant colon tissue, and cells were pelleted by centrifugation at 4000 rpm for 10 minutes at 4C. Supernatant was removed and the cell pellet was resuspended in I mL Tri Reagent(Molecular Research Center) for subsequent RNA and protein extraction. Cytochrome c oxidase activity was evaluated as described before in detail (29) Caco-2 cell culture Caco-2 cells were cultured on l x Minimal Essential Media(Gibco), with 10%Fetal Bovine Serum(gibco) and lx glutamax( Gibco)atat 37C and 5% Co2. The cells were seeded at I x cells per six-well plates overnight prior to experiments. When necessary, cells were pre-treated with rhIL-22(100ng/mL, R&D)and rhIFNy(40 ng/mL, R&D) for 30 minutes nd cytokines(rhIL-22 and rhIFNy ), 2mM sodium butyrate(Sigma-Aldrich), 5mM sodium butyrate(Sigma-Aldrich) or 5uM Rosiglitazone( Cayman) were added to the wells and kept throughout the experiments. At 8 or 20 hours after treatments, cells were collected for gene expression or protein analysis by Western Blot, as described below. For nitrate measureme experiments, a trans well plate model of polarized intestinal cells was used as previously described (30). Briefly, Caco-2 cells were maintained on minimum essential medium (MEM) containing 10% fetal bovine serum(FBS). To polarize Caco-2 cells, 0.5 ml of medium containing approximately 1 x 10>cells was seeded apically in 0.4-um 12-mm Transwell plates(polycarbonate membrane, Corning-Costar ), and 1.0 ml of medium was added to the basolateral compartment. The medium was changed every 2 days, and the transepithelial electrical resistance was measured after a week and the day before the experiment using the Millicell-ERS electrical resistance system. When necessary, cells were pre-treated with rhIL-22 (100ng/mL, R&D)and rhIFNy(40 ng/mL, R&D)for 30 minutes Science Author manuscript; available in PMC 2017 October 1For Treg depletion, mice were intraperitoneally injected with 250 μg/mouse of PC61 mAb or isotype control, as previously described (28). At day 10 after treatment, mice were inoculated with 1 × 109 CFU of a 1:1 mixture of the indicated E. coli or S. Typhimurium strains. Samples were collected at 4 days after infection. For microbiota analysis, mice were euthanized at 3 days after streptomycin treatment, and DNA from the colon contents was extracted using the PowerSoil DNA Isolation kit (Mo￾Bio) according to the manufacturer’s protocol. Generation and analysis of sequencing data is described below. Colonocyte isolation The proximal colon was flushed with ice-cold phosphate buffered saline (PBS) (Gibco) using a 10 mL syringe fitted with an 18-gauge blunt needle before opening lengthwise, cutting into 2–4 cm pieces and placing in a 15 mL conical centrifuge tube filled with 10 mL ice-cold PBS (Gibco) on ice. Tubes were gently inverted four times, PBS (Gibco) wash was removed and replaced with additional ice-cold PBS (Gibco) before transfer to sterile petri dishes where colon tissue was cut into <5 mm pieces using a razor blade. Tissue pieces were placed in crypt chelating buffer (2 mM EDTA, pH 8 in PBS) and incubated for 30 minutes with gentle rocking buried in ice. Chelating buffer was removed and replaced with cold dissociation buffer (54.9 mM D-sorbitol, 43.4 mM sucrose in PBS) before shaking vigorously by hand for 8 minutes. Cell suspension was transferred to a new 15 mL conical centrifuge tube, leaving any large remnant colon tissue, and cells were pelleted by centrifugation at 4000 rpm for 10 minutes at 4°C. Supernatant was removed and the cell pellet was resuspended in 1 mL Tri Reagent (Molecular Research Center) for subsequent RNA and protein extraction. Cytochrome c oxidase activity was evaluated as described before in detail (29). Caco-2 cell culture Caco-2 cells were cultured on 1× Minimal Essential Media (Gibco), with 10% Fetal Bovine Serum (Gibco) and 1× Glutamax (Gibco) at at 37°C and 5% CO2. The cells were seeded at 1 × 105 cells per six-well plates overnight prior to experiments. When necessary, cells were pre-treated with rhIL-22 (100ng/mL, R&D) and rhIFNγ (40 ng/mL, R&D) for 30 minutes and cytokines (rhIL-22 and rhIFNγ), 2mM sodium butyrate (Sigma-Aldrich), 5mM sodium butyrate (Sigma-Aldrich) or 5μM Rosiglitazone (Cayman) were added to the wells and kept throughout the experiments. At 8 or 20 hours after treatments, cells were collected for gene expression or protein analysis by Western Blot, as described below. For nitrate measurement experiments, a trans well plate model of polarized intestinal cells was used as previously described (30). Briefly, Caco-2 cells were maintained on minimum essential medium (MEM) containing 10% fetal bovine serum (FBS). To polarize Caco-2 cells, 0.5 ml of medium containing approximately 1 × 105 cells was seeded apically in 0.4-μm 12-mm Transwell plates (polycarbonate membrane, Corning-Costar), and 1.0 ml of medium was added to the basolateral compartment. The medium was changed every 2 days, and the transepithelial electrical resistance was measured after a week and the day before the experiment using the Millicell-ERS electrical resistance system. When necessary, cells were pre-treated with rhIL-22 (100ng/mL, R&D) and rhIFNγ (40 ng/mL, R&D) for 30 minutes Byndloss et al. Page 9 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript