#METABOLOMICS WORKBENCH flopes_20220701_124837 DATATRACK_ID:3326 STUDY_ID:ST002230 ANALYSIS_ID:AN003639 PROJECT_ID:PR001420 VERSION 1 CREATED_ON July 19, 2022, 10:45 am #PROJECT PR:PROJECT_TITLE Metabolomics of bone marrow-derived dendritic cells conditioned with H. PR:PROJECT_TITLE polygyrus bakery non-polar metabolites PR:PROJECT_SUMMARY Aim: Characterize tolerogenic responses induced by helminth-derived metabolites PR:PROJECT_SUMMARY (HDM) in dendritic cells (DCs). Methods: H. polygyrus worms were culture for 24h PR:PROJECT_SUMMARY and HDMs were isolated from conditioned media by chromatography. Bone PR:PROJECT_SUMMARY marrow-derived dendritic cells (BMDCs) were treated with HDM for 4 or 20 h. PR:PROJECT_SUMMARY Cells were either stimulated with LPS or adoptively transferred to mice. PR:PROJECT_SUMMARY Cytokine secretion was measured by ELISA. The metabolome of HDM-treated DCs were PR:PROJECT_SUMMARY assessed by mass spectrometry, respectively. Results: Pre-treatment with HDM PR:PROJECT_SUMMARY decreased LPS-induced TNF and increased IL-10 release by BMDCs. Importantly, HDM PR:PROJECT_SUMMARY decreased expression of MHC-II, CD86, and CD40 in BMDCs and splenic DCs, PR:PROJECT_SUMMARY suggesting that HDM induces a tolerogenic profile on DCs. The metabolomic PR:PROJECT_SUMMARY approach revealed a total of 17 downregulated metabolites, against one PR:PROJECT_SUMMARY upregulated of the 225 total peaks analyzed. Functional analyses were performed PR:PROJECT_SUMMARY and results predicted a total of 29 pathways and 43 matched compounds. Scatter PR:PROJECT_SUMMARY plot test of significant peaks revealed two differentially enriched pathways, PR:PROJECT_SUMMARY the sphingolipid metabolism, and a highly enriched pathway, the terpenoid PR:PROJECT_SUMMARY backbone metabolism, witch C00418 metabolite is a potential match to mevalonic PR:PROJECT_SUMMARY acid, according to KEGG compound database in HDM-treated DCs in comparison with PR:PROJECT_SUMMARY naïve DCs. These differentially expressed genes and enriched metabolites may PR:PROJECT_SUMMARY indicate a novel mechanism by which helminths induce a tolerogenic profile in PR:PROJECT_SUMMARY DCs. PR:INSTITUTE McGill University PR:LAST_NAME Lopes PR:FIRST_NAME Fernando PR:ADDRESS 21111 Lakeshore Rd PR:EMAIL fernando.lopes@mcgill.ca PR:PHONE 5143987607 #STUDY ST:STUDY_TITLE Metabolomics of bone marrow-derived dendritic cells conditioned with H. ST:STUDY_TITLE polygyrus bakery non-polar metabolites ST:STUDY_SUMMARY Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. ST:STUDY_SUMMARY polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of ST:STUDY_SUMMARY 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF ST:STUDY_SUMMARY at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were ST:STUDY_SUMMARY collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, ST:STUDY_SUMMARY centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until ST:STUDY_SUMMARY analysis. The profiling of nonpolar metabolites was performed by LC-MS/MS ST:STUDY_SUMMARY analysis of the deproteinated conditioned media by injecting 3 mL of sample onto ST:STUDY_SUMMARY a Dionex UHPLC system equipped with an Agilent Eclipse C18 (2.1 x 15 mm, 1.8 mm) ST:STUDY_SUMMARY column incubated at 45oC. Metabolites were resolved with a 30 min linear running ST:STUDY_SUMMARY 0-80 % using the buffers system 0.05 % formic acid and 0.05 % formic acid in ST:STUDY_SUMMARY acetonitrile at a flowrate of 300 mL/min. The column effluent was introduced by ST:STUDY_SUMMARY electrospray ionization onto a ThermoScientific Velos LTQ Orbitrap Analyzer ST:STUDY_SUMMARY using a spray voltage of 3.6 kV, a source heater temperature of 350oC, and a ST:STUDY_SUMMARY sheath gas flow of 40 L/min. Survey scans were performed using the Orbitrap mass ST:STUDY_SUMMARY spectrometer and the 10 most intense ions were selected for fragmentation using ST:STUDY_SUMMARY a 30-40 V stepped collision induced dissociation energy. Fragmentation products ST:STUDY_SUMMARY were analyzed in the linear ion trap mass spectrometer. Fragmentation was used ST:STUDY_SUMMARY to perform XCMS online database (https://xcmsonline.scripps.edu) search to ST:STUDY_SUMMARY identify possible metabolites. ST:INSTITUTE McGill University ST:LAST_NAME Lopes ST:FIRST_NAME Fernando ST:ADDRESS 21111 Lakeshore Rd ST:EMAIL fernando.lopes@mcgill.ca ST:PHONE 5143987607 #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]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - FLmedia_1 Experimental variables:Cell-free media RAW_FILE_NAME=FLmedia_1.raw SUBJECT_SAMPLE_FACTORS - FLmedia_2 Experimental variables:Cell-free media RAW_FILE_NAME=FLmedia_2.raw SUBJECT_SAMPLE_FACTORS - FLmedia_3 Experimental variables:Cell-free media RAW_FILE_NAME=FLmedia_3.raw SUBJECT_SAMPLE_FACTORS - FLcontrol20h_1 Experimental variables:Supernatant of DCs negative control cultured for 20h RAW_FILE_NAME=FLcontrol20h_1.raw SUBJECT_SAMPLE_FACTORS - FLcontrol20h_2 Experimental variables:Supernatant of DCs negative control cultured for 20h RAW_FILE_NAME=FLcontrol20h_2.raw SUBJECT_SAMPLE_FACTORS - FLcontrol20h_3 Experimental variables:Supernatant of DCs negative control cultured for 20h RAW_FILE_NAME=FLcontrol20h_3.raw SUBJECT_SAMPLE_FACTORS - FLcontrol20h_4 Experimental variables:Supernatant of DCs negative control cultured for 20h RAW_FILE_NAME=FLcontrol20h_4.raw SUBJECT_SAMPLE_FACTORS - FLControl4h_1 Experimental variables:Supernatant of DCs negative control cultured for 4h RAW_FILE_NAME=FLControl4h_1.raw SUBJECT_SAMPLE_FACTORS - FLControl4h_2 Experimental variables:Supernatant of DCs negative control cultured for 4h RAW_FILE_NAME=FLControl4h_2.raw SUBJECT_SAMPLE_FACTORS - FLControl4h_3 Experimental variables:Supernatant of DCs negative control cultured for 4h RAW_FILE_NAME=FLControl4h_3.raw SUBJECT_SAMPLE_FACTORS - FLHpb20h_1 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h RAW_FILE_NAME=FLHpb20h_1.raw SUBJECT_SAMPLE_FACTORS - FLHpb20h_2 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h RAW_FILE_NAME=FLHpb20h_2.raw SUBJECT_SAMPLE_FACTORS - FLHpb20h_3 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h RAW_FILE_NAME=FLHpb20h_3.raw SUBJECT_SAMPLE_FACTORS - FLHpb20h_4 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h RAW_FILE_NAME=FLHpb20h_4.raw SUBJECT_SAMPLE_FACTORS - Hpb4h_1 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h RAW_FILE_NAME=Hpb4h_1.raw SUBJECT_SAMPLE_FACTORS - Hpb4h_2 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h RAW_FILE_NAME=Hpb4h_2.raw SUBJECT_SAMPLE_FACTORS - Hpb4h_3 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h RAW_FILE_NAME=Hpb4h_3.raw SUBJECT_SAMPLE_FACTORS - Hpb4h_4 Experimental variables:Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h RAW_FILE_NAME=Hpb4h_4.raw #COLLECTION CO:COLLECTION_SUMMARY Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. CO:COLLECTION_SUMMARY polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of CO:COLLECTION_SUMMARY 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF CO:COLLECTION_SUMMARY at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were CO:COLLECTION_SUMMARY collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, CO:COLLECTION_SUMMARY centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until CO:COLLECTION_SUMMARY analysis. CO:COLLECTION_PROTOCOL_FILENAME Summary_of_the_study_protocols.docx CO:SAMPLE_TYPE Dendritic cells #TREATMENT TR:TREATMENT_SUMMARY Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. TR:TREATMENT_SUMMARY polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of TR:TREATMENT_SUMMARY 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF TR:TREATMENT_SUMMARY at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were TR:TREATMENT_SUMMARY collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, TR:TREATMENT_SUMMARY centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until TR:TREATMENT_SUMMARY analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. SP:SAMPLEPREP_SUMMARY polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of SP:SAMPLEPREP_SUMMARY 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF SP:SAMPLEPREP_SUMMARY at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were SP:SAMPLEPREP_SUMMARY collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, SP:SAMPLEPREP_SUMMARY centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until SP:SAMPLEPREP_SUMMARY analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME Summary_of_the_study_protocols.docx #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex CH:COLUMN_NAME Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) CH:METHODS_FILENAME Summary_of_the_study_protocols.docx #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Summary_of_the_study_protocols.docx #MS MS:INSTRUMENT_NAME Thermo LTQ Discovery Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Fragmentation products were analyzed in the linear ion trap mass spectrometer. MS:MS_COMMENTS Fragmentation was used to perform XCMS online database MS:MS_COMMENTS (https://xcmsonline.scripps.edu) search to identify possible metabolites. MS:MS_RESULTS_FILE ST002230_AN003639_Results.txt UNITS:M/z Has m/z:Yes Has RT:Yes RT units:Minutes #END