Bindloss et al and cytokines(rhlL-22 and rhIFNg), 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 24 hours after treatments, supernatants from each well were collected and immediately used for nitrate measurements as described below. All experiments were repeated at least four times independently Real time Pcr RNA from caco-2 cells or colonocytes was isolated with Tri-reagent(Molecular Research Center) according to the instructions of the manufacturer. A reverse transcriptase reaction was performed to prepare complementary DNA(cDNA)using TaqMan reverse transcription reagents(Applied Biosystems). A volume of 4 ul of cDNA was used as template for each eal-time PCR reaction in a total reaction volume of 25 uL. Real-time PCR was performed using SYBR Green(Applied Biosystems). Data were analyzed using the comparative Ct method(Applied Biosystems). Transcript levels of Nos2 and were normalized to mRNA levels of the housekeeping gene Act2b, encoding B-actin Short-chain fatty acid measurement Cecum content samples were weighed, diluted with nanopure water to I mg/10 uL, and homogenized in the shaker overnight. The samples were then centrifuged at 21130xg for 20 minutes, and 20uL of supernatant was mixed with 20 HL 0. 2 M 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride in 5% pyridine/water(v/v)and 40 uL 0.1 M 2-nitrophenylhydrazine in 80% ACN/water(v/v)with 0.05 M HCl. The mixture was allowed to react at 40C for 30 min and cool to room temperature. Then 400 uL of 10% ACN/water(v/v)was added. The solution was centrifuged at 4000xg for 30 minutes. The supernatant was injected to ultra high performance liquid chromatography triple quadrupole mass spectrometer(UHPLC-QqQ-MS)for short chain fatty acid analysis Microbiota analysis Primers 515F (AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTATGGTAATTGTGTGCCA GCMGC CGCGGTAA)and 806R (CAAGCAGAAGACGGCATACGAGATNNNNNNNNAGTCAGTCAGCCGGACTACHV GGGWTC TAAT) were used to amplify the v4 domain of the bacterial 16S rRNA. Both forward and reverse primers contained a unique 8 nt barcode(N), a primer pad, a linker sequence, and the Illumina adaptor sequences. Each sample was barcoded with a unique forward and reverse barcode combination. PCR contained I Unit Kapa2G Robust Hot Start Polymerase(Kapa Biosystems), 1.5 mM MgCl2, 10 pmol of each primer and lul of dNA PCR conditions were: an initial incubation at 95.C for 2 min, followed by 30 cycles of 95C for 10s. 55%C for 15s. 72oC for 15 s and a final extension of 72C for 3 min The final product was quantified on the Qubit instrument using the Qubit High Sensitivity DNA kit (Invitrogen )and individual amplicon libraries were pooled, cleaned by Ampure XP beads (Beckman Coulter), and sequenced using a 250 bp paired-end method on an Illumina MiSeq instrument in the Genome Center DNA Technologies Core, University of California, Davis Qiimeopen-sourcesoftware(http://qiime.org)(caporasoetal.,2010)wasusedforinitial identification of operational taxonomic units(OTD), clustering, and phylogenetic analysis Science Author manuscript; available in PMC 2017 October 1and cytokines (rhIL-22 and rhIFNg), 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 24 hours after treatments, supernatants from each well were collected and immediately used for nitrate measurements as described below. All experiments were repeated at least four times independently. Real time PCR RNA from caco-2 cells or colonocytes was isolated with Tri-reagent (Molecular Research Center) according to the instructions of the manufacturer. A reverse transcriptase reaction was performed to prepare complementary DNA (cDNA) using TaqMan reverse transcription reagents (Applied Biosystems). A volume of 4 μl of cDNA was used as template for each real-time PCR reaction in a total reaction volume of 25 μL. Real-time PCR was performed using SYBR Green (Applied Biosystems). Data were analyzed using the comparative Ct method (Applied Biosystems). Transcript levels of Nos2 and were normalized to mRNA levels of the housekeeping gene Act2b, encoding β-actin. Short-chain fatty acid measurements Cecum content samples were weighed, diluted with nanopure water to 1 mg/10 μL, and homogenized in the shaker overnight. The samples were then centrifuged at 21130×g for 20 minutes, and 20μL of supernatant was mixed with 20 μL 0.2 M 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride in 5% pyridine/water (v/v) and 40 μL 0.1 M 2-nitrophenylhydrazine in 80% ACN/water (v/v) with 0.05 M HCl. The mixture was allowed to react at 40 °C for 30 min and cool to room temperature. Then 400 μL of 10% ACN/water (v/v) was added. The solution was centrifuged at 4000×g for 30 minutes. The supernatant was injected to ultra high performance liquid chromatography triple quadrupole mass spectrometer (UHPLC-QqQ-MS) for short chain fatty acid analysis. Microbiota analysis Primers 515F (AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTATGGTAATTGTGTGCCA GCMGC CGCGGTAA) and 806R (CAAGCAGAAGACGGCATACGAGATNNNNNNNNAGTCAGTCAGCCGGACTACHV GGGWTC TAAT) were used to amplify the V4 domain of the bacterial 16S rRNA. Both forward and reverse primers contained a unique 8 nt barcode (N), a primer pad, a linker sequence, and the Illumina adaptor sequences. Each sample was barcoded with a unique forward and reverse barcode combination. PCR contained 1 Unit Kapa2G Robust Hot Start Polymerase (Kapa Biosystems), 1.5 mM MgCl2, 10 pmol of each primer and 1ul of DNA. PCR conditions were: an initial incubation at 95°C for 2 min, followed by 30 cycles of 95°C for 10 s, 55°C for 15 s, 72°C for 15 s and a final extension of 72°C for 3 min. The final product was quantified on the Qubit instrument using the Qubit High Sensitivity DNA kit (Invitrogen) and individual amplicon libraries were pooled, cleaned by Ampure XP beads (Beckman Coulter), and sequenced using a 250 bp paired-end method on an Illumina MiSeq instrument in the Genome Center DNA Technologies Core, University of California, Davis. Qiime open-source software (http://qiime.org) (Caporaso et al., 2010) was used for initial identification of operational taxonomic units (OTU), clustering, and phylogenetic analysis. Byndloss et al. Page 10 Science. Author manuscript; available in PMC 2017 October 16. Author Manuscript Author Manuscript Author Manuscript Author Manuscript