#METABOLOMICS WORKBENCH rgn5011_20190719_074408 DATATRACK_ID:1775 STUDY_ID:ST001219 ANALYSIS_ID:AN002032 PROJECT_ID:PR000818 VERSION 1 CREATED_ON July 19, 2019, 6:57 pm #PROJECT PR:PROJECT_TITLE Vitamin D regulates the microbiota to induce RORgt/FoxP3+ regulatory T cells PR:PROJECT_SUMMARY The active form of vitamin D (1,25(OH)2D) suppresses experimental models of PR:PROJECT_SUMMARY inflammatory bowel disease in part by regulating the microbiota. In this study, PR:PROJECT_SUMMARY the role of vitamin D in the regulation of microbe induced RORgt/FoxP3+ T PR:PROJECT_SUMMARY regulatory (reg) cells in the colon was determined. Vitamin D sufficient (D+) PR:PROJECT_SUMMARY mice had significantly higher frequencies of FoxP3+ and RORgt/FoxP3+ T reg cells PR:PROJECT_SUMMARY in the colon compared to vitamin D deficient (D-) mice. The higher frequency of PR:PROJECT_SUMMARY RORgt/FoxP3+ T reg cells in D+ colon correlated with higher numbers of bacteria PR:PROJECT_SUMMARY from the Clostridium XIVa and Bacteroides in D+ compared to D- cecum. D- mice PR:PROJECT_SUMMARY with fewer RORgt/FoxP3+ T reg cells were significantly more susceptible to PR:PROJECT_SUMMARY colitis than D+ mice. Transfer of the cecal bacteria from D+ or D- mice to PR:PROJECT_SUMMARY germfree recipients phenocopied the higher numbers of RORgt/FoxP3+ cells and PR:PROJECT_SUMMARY reduced susceptibility to colitis in D+ versus D- recipient mice. 1,25(OH)2D PR:PROJECT_SUMMARY treatment of the D- mice beginning at 3 weeks of age did not completely recover PR:PROJECT_SUMMARY RORgt/FoxP3+ T reg cells or the Bacteriodes, Bacteriodes thetaiotaomicron, and PR:PROJECT_SUMMARY Clostridium XIVa numbers to D+ values. Early vitamin D status shapes the PR:PROJECT_SUMMARY microbiota to optimize the population of colonic RORgt/FoxP3+ T reg cells PR:PROJECT_SUMMARY important for resistance to colitis. PR:INSTITUTE The Pennsylvania State University (Penn State) PR:LAST_NAME Nichols PR:FIRST_NAME Robert PR:ADDRESS 650 toftrees Ave apt 108, state college, PA, 16803 PR:EMAIL rgn5011@psu.edu PR:PHONE 7247662694 #STUDY ST:STUDY_TITLE Vitamin D regulates the microbiota to induce RORgt/FoxP3+ regulatory T cells ST:STUDY_SUMMARY The active form of vitamin D (1,25(OH)2D) suppresses experimental models of ST:STUDY_SUMMARY inflammatory bowel disease in part by regulating the microbiota. In this study, ST:STUDY_SUMMARY the role of vitamin D in the regulation of microbe induced RORgt/FoxP3+ T ST:STUDY_SUMMARY regulatory (reg) cells in the colon was determined. Vitamin D sufficient (D+) ST:STUDY_SUMMARY mice had significantly higher frequencies of FoxP3+ and RORgt/FoxP3+ T reg cells ST:STUDY_SUMMARY in the colon compared to vitamin D deficient (D-) mice. The higher frequency of ST:STUDY_SUMMARY RORgt/FoxP3+ T reg cells in D+ colon correlated with higher numbers of bacteria ST:STUDY_SUMMARY from the Clostridium XIVa and Bacteroides in D+ compared to D- cecum. D- mice ST:STUDY_SUMMARY with fewer RORgt/FoxP3+ T reg cells were significantly more susceptible to ST:STUDY_SUMMARY colitis than D+ mice. Transfer of the cecal bacteria from D+ or D- mice to ST:STUDY_SUMMARY germfree recipients phenocopied the higher numbers of RORgt/FoxP3+ cells and ST:STUDY_SUMMARY reduced susceptibility to colitis in D+ versus D- recipient mice. 1,25(OH)2D ST:STUDY_SUMMARY treatment of the D- mice beginning at 3 weeks of age did not completely recover ST:STUDY_SUMMARY RORgt/FoxP3+ T reg cells or the Bacteriodes, Bacteriodes thetaiotaomicron, and ST:STUDY_SUMMARY Clostridium XIVa numbers to D+ values. Early vitamin D status shapes the ST:STUDY_SUMMARY microbiota to optimize the population of colonic RORgt/FoxP3+ T reg cells ST:STUDY_SUMMARY important for resistance to colitis. ST:INSTITUTE The Pennsylvania State University (Penn State) ST:LAST_NAME Nichols ST:FIRST_NAME Robert ST:ADDRESS 917 Old Boalsburg Road, State College, Pennsylvania, 16801, USA ST:EMAIL rgn5011@psu.edu ST:PHONE 7247662694 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-1 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-2 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-3 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-4 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-5 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-6 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-7 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-8 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-9 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-10 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-11 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-12 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-13 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-14 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-15 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-16 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-17 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-18 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-19 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-20 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-21 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-22 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-23 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-24 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-25 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-26 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-27 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4A-28 Group:Vitamin D- SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-1 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-2 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-3 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-4 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-5 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-6 Group:Vitamin D - SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-7 Group:Vitamin D - SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-8 Group:Vitamin D - SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-9 Group:Vitamin D - SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-10 Group:Vitamin D - SUBJECT_SAMPLE_FACTORS - Sup_Fig4B-11 Group:Vitamin D - SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-1 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-2 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-3 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-4 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-5 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-6 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-7 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-8 Group:Vitamin D+ SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-9 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-10 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-11 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-12 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-13 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-14 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-15 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-16 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-17 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-18 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-19 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-20 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-21 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-22 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-23 Group:Vitamin D-, +1,25D SUBJECT_SAMPLE_FACTORS - Sup_Fig4C-24 Group:Vitamin D-, +1,25D #COLLECTION CO:COLLECTION_SUMMARY C57BL/6 mice were originally from Jackson Labs (Bar Harbor, MN) and maintained CO:COLLECTION_SUMMARY at the Pennsylvania State University (University Park, PA). All mice used were CO:COLLECTION_SUMMARY housed within the same rooms in the animal facility. Mice were fed purified CO:COLLECTION_SUMMARY diets made in the lab as described that either contained vitamin D (D+) or did CO:COLLECTION_SUMMARY not (D-) 24. GF C57BL/6 mice were bred and maintained at the Pennsylvania State CO:COLLECTION_SUMMARY University gnotobiotic animal research facility. For some experiments, 1,25D was CO:COLLECTION_SUMMARY started in the diets of 3-5 week old mice (25ng/d until 5 weeks of age) and mice CO:COLLECTION_SUMMARY that were >5 weeks old were fed 50ng/d 1,25D exactly as described 24. For CO:COLLECTION_SUMMARY microbial transplantation experiments (two independent experiments), the 4 week CO:COLLECTION_SUMMARY old GF mice were gavaged with 100 µl/10 µg cecal contents from D+ and D- mice, CO:COLLECTION_SUMMARY and used for experiments after 2 weeks 25. All of the experimental procedures CO:COLLECTION_SUMMARY were approved by the Institutional Animal Care and Use Committee at the CO:COLLECTION_SUMMARY Pennsylvania State University CO:SAMPLE_TYPE Cecum #TREATMENT TR:TREATMENT_SUMMARY C57BL/6 mice were originally from Jackson Labs (Bar Harbor, MN) and maintained TR:TREATMENT_SUMMARY at the Pennsylvania State University (University Park, PA). All mice used were TR:TREATMENT_SUMMARY housed within the same rooms in the animal facility. Mice were fed purified TR:TREATMENT_SUMMARY diets made in the lab as described that either contained vitamin D (D+) or did TR:TREATMENT_SUMMARY not (D-) 24. GF C57BL/6 mice were bred and maintained at the Pennsylvania State TR:TREATMENT_SUMMARY University gnotobiotic animal research facility. For some experiments, 1,25D was TR:TREATMENT_SUMMARY started in the diets of 3-5 week old mice (25ng/d until 5 weeks of age) and mice TR:TREATMENT_SUMMARY that were >5 weeks old were fed 50ng/d 1,25D exactly as described 24. For TR:TREATMENT_SUMMARY microbial transplantation experiments (two independent experiments), the 4 week TR:TREATMENT_SUMMARY old GF mice were gavaged with 100 µl/10 µg cecal contents from D+ and D- mice, TR:TREATMENT_SUMMARY and used for experiments after 2 weeks 25. All of the experimental procedures TR:TREATMENT_SUMMARY were approved by the Institutional Animal Care and Use Committee at the TR:TREATMENT_SUMMARY Pennsylvania State University #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Extract metabolites from cecal contents, then run samples. Pleas see the NMR SP:SAMPLEPREP_SUMMARY protocol doc attached to this file #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Bruker Avance II NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 600 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS umol/mg NMR_METABOLITE_DATA_START Samples Sup_Fig4A-1 Sup_Fig4A-2 Sup_Fig4A-3 Sup_Fig4A-4 Sup_Fig4A-5 Sup_Fig4A-6 Sup_Fig4A-7 Sup_Fig4A-8 Sup_Fig4A-9 Sup_Fig4A-10 Sup_Fig4A-11 Sup_Fig4A-12 Sup_Fig4A-13 Sup_Fig4A-14 Sup_Fig4A-15 Sup_Fig4A-16 Sup_Fig4A-17 Sup_Fig4A-18 Sup_Fig4A-19 Sup_Fig4A-20 Sup_Fig4A-21 Sup_Fig4A-22 Sup_Fig4A-23 Sup_Fig4A-24 Sup_Fig4A-25 Sup_Fig4A-26 Sup_Fig4A-27 Sup_Fig4A-28 Sup_Fig4B-1 Sup_Fig4B-2 Sup_Fig4B-3 Sup_Fig4B-4 Sup_Fig4B-5 Sup_Fig4B-6 Sup_Fig4B-7 Sup_Fig4B-8 Sup_Fig4B-9 Sup_Fig4B-10 Sup_Fig4B-11 Sup_Fig4C-1 Sup_Fig4C-2 Sup_Fig4C-3 Sup_Fig4C-4 Sup_Fig4C-5 Sup_Fig4C-7 Sup_Fig4C-8 Sup_Fig4C-9 Sup_Fig4C-10 Sup_Fig4C-11 Sup_Fig4C-12 Sup_Fig4C-13 Sup_Fig4C-14 Sup_Fig4C-15 Sup_Fig4C-16 Sup_Fig4C-17 Sup_Fig4C-18 Sup_Fig4C-19 Sup_Fig4C-20 Sup_Fig4C-21 Sup_Fig4C-22 Sup_Fig4C-23 Sup_Fig4C-24 Factors Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D- Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D - Group:Vitamin D - Group:Vitamin D - Group:Vitamin D - Group:Vitamin D - Group:Vitamin D - Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D+ Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Group:Vitamin D-, +1,25D Acetate 0.092018 0.04724 0.047775 0.057088 0.055718 0.064326 0.082945 0.075789 0.057116 0.055129 0.09863 0.207109 0.207294 0.121225 0.154115 0.125352 0.205691 0.14637 0.172493 0.147522 0.173166 0.173455 0.171588 0.174626 0.103454 0.158273 0.125274 0.162361 0.195676 0.153066 0.13909 0.181311 0.130654 0.169576 Propionate 0.02236919 0.02161789 0.01597122 0.0150247 0.02149741 0.01656917 0.02164688 0.01803277 0.02979923 0.02824199 0.02013089 0.02104063 0.02201613 0.02494664 0.02906941 0.01689555 0.01416869 0.01687083 0.02002514 0.02318754 0.01953432 0.02141102 0.0201564 0.02256287 0.01753138 0.02344396 0.01502563 0.01730252 0.005674 0.002562 0.003301 0.003487 0.003404 0.006062 0.007078 0.00521 0.005587 0.005051 0.007536 0.023023 0.056309 0.025937 0.028748 0.029073 0.055092 0.036067 0.036666 0.025206 0.041513 0.032802 0.04062 0.023482 0.017415 0.025577 0.022599 0.028587 0.033419 0.028431 0.027795 0.025909 0.025628 0.02823 Butyrate 0.017202 0.007609 0.009134 0.007954 0.008572 0.007289 0.011329 0.00966 0.009169 0.008651 0.013806 0.013807 0.009438 0.007269 0.013056 0.012837 0.013038 0.008807 0.018597 0.010756 0.014247 0.017011 0.013368 0.011599 0.009121 0.015941 0.018193 0.010265 0.02271 0.010538 0.009106 0.018916 0.006468 0.012187 Total SCFA 0.243939 0.273042 0.154431 0.195919 0.167262 0.273821 0.191243 0.227756 0.183485 0.228927 0.223269 0.225577 0.209707 0.12999 0.199792 0.166065 0.201213 0.251805 0.192035 0.17599 0.226137 0.16275 0.209992 bile acids 0.00155541 0.00130299 0.01130576 0.00747897 0.00573008 0.00745525 0.00314569 0.0069039 0.01366912 0.00887538 0.01057139 0.01570512 0.01428016 0.00586194 0.00919269 0.00895696 0.00793012 0.00538422 0.00434079 0.01128512 0.00469732 0.00671893 0.00705322 0.00705189 0.00168222 0.00216882 0.00508116 0.00594501 5-aminovalerate 0.02469565 0.01973023 0.01261252 0.00929949 0.0108113 0.01075488 0.01962241 0.01421958 0.01256614 0.01266062 0.01970457 0.02440056 0.02826522 0.02796685 0.0275351 0.02848566 0.03087532 0.02779217 0.00946463 0.01038812 0.01115949 0.01072566 0.0104522 0.01144169 0.01091271 0.00949698 0.01218139 0.01051873 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Type Acetate SCFA Propionate SCFA Butyrate SCFA Total SCFA SCFA bile acids Bile acid 5-aminovalerate 5-aminovalerate METABOLITES_END #END