{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002230","ANALYSIS_ID":"AN003639","VERSION":"1","CREATED_ON":"July 19, 2022, 10:45 am"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics of bone marrow-derived dendritic cells conditioned with H. polygyrus bakery non-polar metabolites","PROJECT_SUMMARY":"Aim: Characterize tolerogenic responses induced by helminth-derived metabolites (HDM) in dendritic cells (DCs). Methods: H. polygyrus worms were culture for 24h and HDMs were isolated from conditioned media by chromatography. Bone marrow-derived dendritic cells (BMDCs) were treated with HDM for 4 or 20 h. Cells were either stimulated with LPS or adoptively transferred to mice. Cytokine secretion was measured by ELISA. The metabolome of HDM-treated DCs were assessed by mass spectrometry, respectively. Results: Pre-treatment with HDM decreased LPS-induced TNF and increased IL-10 release by BMDCs. Importantly, HDM decreased expression of MHC-II, CD86, and CD40 in BMDCs and splenic DCs, suggesting that HDM induces a tolerogenic profile on DCs. The metabolomic approach revealed a total of 17 downregulated metabolites, against one upregulated of the 225 total peaks analyzed. Functional analyses were performed and results predicted a total of 29 pathways and 43 matched compounds. Scatter plot test of significant peaks revealed two differentially enriched pathways, the sphingolipid metabolism, and a highly enriched pathway, the terpenoid backbone metabolism, witch C00418 metabolite is a potential match to mevalonic acid, according to KEGG compound database in HDM-treated DCs in comparison with naïve DCs. These differentially expressed genes and enriched metabolites may indicate a novel mechanism by which helminths induce a tolerogenic profile in DCs.","INSTITUTE":"McGill University","LAST_NAME":"Lopes","FIRST_NAME":"Fernando","ADDRESS":"21111 Lakeshore Rd","EMAIL":"fernando.lopes@mcgill.ca","PHONE":"5143987607"},

"STUDY":{"STUDY_TITLE":"Metabolomics of bone marrow-derived dendritic cells conditioned with H. polygyrus bakery non-polar metabolites","STUDY_SUMMARY":"Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until analysis. The profiling of nonpolar metabolites was performed by LC-MS/MS analysis of the deproteinated conditioned media by injecting 3 mL of sample onto a Dionex UHPLC system equipped with an Agilent Eclipse C18 (2.1 x 15 mm, 1.8 mm) column incubated at 45oC. Metabolites were resolved with a 30 min linear running 0-80 % using the buffers system 0.05 % formic acid and 0.05 % formic acid in acetonitrile at a flowrate of 300 mL/min. The column effluent was introduced by electrospray ionization onto a ThermoScientific Velos LTQ Orbitrap Analyzer using a spray voltage of 3.6 kV, a source heater temperature of 350oC, and a sheath gas flow of 40 L/min. Survey scans were performed using the Orbitrap mass spectrometer and the 10 most intense ions were selected for fragmentation using a 30-40 V stepped collision induced dissociation energy. Fragmentation products were analyzed in the linear ion trap mass spectrometer. Fragmentation was used to perform XCMS online database (https://xcmsonline.scripps.edu) search to identify possible metabolites.","INSTITUTE":"McGill University","LAST_NAME":"Lopes","FIRST_NAME":"Fernando","ADDRESS":"21111 Lakeshore Rd","EMAIL":"fernando.lopes@mcgill.ca","PHONE":"5143987607"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"FLmedia_1",
"Factors":{"Experimental variables":"Cell-free media"},
"Additional sample data":{"RAW_FILE_NAME":"FLmedia_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLmedia_2",
"Factors":{"Experimental variables":"Cell-free media"},
"Additional sample data":{"RAW_FILE_NAME":"FLmedia_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLmedia_3",
"Factors":{"Experimental variables":"Cell-free media"},
"Additional sample data":{"RAW_FILE_NAME":"FLmedia_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLcontrol20h_1",
"Factors":{"Experimental variables":"Supernatant of DCs negative control cultured for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLcontrol20h_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLcontrol20h_2",
"Factors":{"Experimental variables":"Supernatant of DCs negative control cultured for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLcontrol20h_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLcontrol20h_3",
"Factors":{"Experimental variables":"Supernatant of DCs negative control cultured for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLcontrol20h_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLcontrol20h_4",
"Factors":{"Experimental variables":"Supernatant of DCs negative control cultured for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLcontrol20h_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLControl4h_1",
"Factors":{"Experimental variables":"Supernatant of DCs negative control cultured for 4h"},
"Additional sample data":{"RAW_FILE_NAME":"FLControl4h_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLControl4h_2",
"Factors":{"Experimental variables":"Supernatant of DCs negative control cultured for 4h"},
"Additional sample data":{"RAW_FILE_NAME":"FLControl4h_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLControl4h_3",
"Factors":{"Experimental variables":"Supernatant of DCs negative control cultured for 4h"},
"Additional sample data":{"RAW_FILE_NAME":"FLControl4h_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLHpb20h_1",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLHpb20h_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLHpb20h_2",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLHpb20h_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLHpb20h_3",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLHpb20h_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"FLHpb20h_4",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 20h"},
"Additional sample data":{"RAW_FILE_NAME":"FLHpb20h_4.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Hpb4h_1",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h"},
"Additional sample data":{"RAW_FILE_NAME":"Hpb4h_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Hpb4h_2",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h"},
"Additional sample data":{"RAW_FILE_NAME":"Hpb4h_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Hpb4h_3",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h"},
"Additional sample data":{"RAW_FILE_NAME":"Hpb4h_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Hpb4h_4",
"Factors":{"Experimental variables":"Supernatant of DCs treated with H. polygyrus bakery non-polar metabolites for 4h"},
"Additional sample data":{"RAW_FILE_NAME":"Hpb4h_4.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until analysis.","COLLECTION_PROTOCOL_FILENAME":"Summary_of_the_study_protocols.docx","SAMPLE_TYPE":"Dendritic cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until analysis."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until analysis.","SAMPLEPREP_PROTOCOL_FILENAME":"Summary_of_the_study_protocols.docx"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex","COLUMN_NAME":"Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um)","METHODS_FILENAME":"Summary_of_the_study_protocols.docx"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"Summary_of_the_study_protocols.docx"},

"MS":{"INSTRUMENT_NAME":"Thermo LTQ Discovery Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Fragmentation products were analyzed in the linear ion trap mass spectrometer. Fragmentation was used to perform XCMS online database (https://xcmsonline.scripps.edu) search to identify possible metabolites.","MS_RESULTS_FILE":"ST002230_AN003639_Results.txt UNITS:M/z Has m/z:Yes Has RT:Yes RT units:Minutes"}

}