{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002378","ANALYSIS_ID":"AN003876","VERSION":"1","CREATED_ON":"12-15-2022"},

"PROJECT":{"PROJECT_TITLE":"Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation","PROJECT_TYPE":"Basic research","PROJECT_SUMMARY":"Activating macrophage NLRP3 inflammasome can promote excessive inflammation, with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves cristae ultrastructure, and attenuates mitochondrial ROS production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. We suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 inflammasome activation in acutely inflamed macrophages.","INSTITUTE":"Wake Forest School of Medicine","LAST_NAME":"Zhu","FIRST_NAME":"Xuewei","ADDRESS":"575 Patterson Ave","EMAIL":"xwzhu@wakehealth.edu","PHONE":"3367131445","DOI":"http://dx.doi.org/10.21228/M8Q13W"},

"STUDY":{"STUDY_TITLE":"Targeted metabolomics analysis of WT and GSDMDKO macrophages","STUDY_TYPE":"MS analysis","STUDY_SUMMARY":"Activating macrophage NLRP3 inflammasome can promote excessive inflammation, with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves cristae ultrastructure, and attenuates mitochondrial ROS production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. We suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 inflammasome activation in acutely inflamed macrophages.","INSTITUTE":"Wake Forest School of Medicine","LAST_NAME":"Zhu","FIRST_NAME":"Xuewei","ADDRESS":"575 Patterson Ave, Winston-Salem, NC 27101","EMAIL":"xwzhu@wakehealth.edu","PHONE":"3367131445","SUBMIT_DATE":"2022-11-16"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENDER":"Male and female"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"GSDMD-LPS-1",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-LPS-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-LPS-2",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-LPS-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-LPS-3",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-LPS-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-LPS-4",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-LPS-4.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-ATP-1",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-ATP-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-ATP-2",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-ATP-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-ATP-3",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-ATP-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-ATP-4",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-ATP-4.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-JX-1",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-JX-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-JX-2",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-JX-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-JX-3",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-JX-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-JX-4",
"Factors":{"Genotype":"GSDMDKO","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-JX-4.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-control-1",
"Factors":{"Genotype":"GSDMDKO","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-control-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-control-2",
"Factors":{"Genotype":"GSDMDKO","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-control-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-control-3",
"Factors":{"Genotype":"GSDMDKO","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-control-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"GSDMD-control-4",
"Factors":{"Genotype":"GSDMDKO","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"GSDMD-control-4.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-LPS-1",
"Factors":{"Genotype":"WT","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"WT-LPS-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-LPS-2",
"Factors":{"Genotype":"WT","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"WT-LPS-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-LPS-3",
"Factors":{"Genotype":"WT","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"WT-LPS-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-LPS-4",
"Factors":{"Genotype":"WT","Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"WT-LPS-4.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-ATP-1",
"Factors":{"Genotype":"WT","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-ATP-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-ATP-2",
"Factors":{"Genotype":"WT","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-ATP-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-ATP-3",
"Factors":{"Genotype":"WT","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-ATP-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-ATP-4",
"Factors":{"Genotype":"WT","Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-ATP-4.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-JX-1",
"Factors":{"Genotype":"WT","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-JX-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-JX-2",
"Factors":{"Genotype":"WT","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-JX-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-JX-3",
"Factors":{"Genotype":"WT","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-JX-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-JX-4",
"Factors":{"Genotype":"WT","Treatment":"LPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"WT-JX-4.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-control-1",
"Factors":{"Genotype":"WT","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"WT-control-1.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-control-2",
"Factors":{"Genotype":"WT","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"WT-control-2.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-control-3",
"Factors":{"Genotype":"WT","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"WT-control-3.mzml"}
},
{
"Subject ID":"-",
"Sample ID":"WT-control-4",
"Factors":{"Genotype":"WT","Treatment":"no treatment"},
"Additional sample data":{"RAW_FILE_NAME":"WT-control-4.mzml"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). Briefly, After treatment, immediately aspirate medium at room temperature. Immediately place the plate on dry ice, and add 1 mL 80% methanol/water (both HPLC grade) (pre-cooled in -80oC for at least 1hr).Remove the plate from -80oC freezer and put it on dry ice, scrape cells into extraction solvent. Transfer the whole cell extract to a new Eppendorf tube placed on ice. Centrifuge at 20 000 rcf for 10 min, 4oC.Transfer the supernatant into two tubes and dry with a speed vacuum at room temperature.","SAMPLE_TYPE":"Macrophages","STORAGE_CONDITIONS":"Described in summary"},

"TREATMENT":{"TREATMENT_SUMMARY":"WT and GSDMD KO bone marrow derived macrophages were first primed with 300 ng/ml LPS (E. coli 0111; B4, Sigma-Aldrich) before stimulated with or without 5 mM ATP (Sigma-Aldrich) for 30 min in the presence or absence of 10 uM JX06. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves cristae ultrastructure, and attenuates mitochondrial ROS production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. We suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 inflammasome activation in acutely inflamed macrophages.","TREATMENT":"In vitro culture","TREATMENT_COMPOUND":"LPS, ATP, JX06"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). Briefly, After treatment, immediately aspirate medium at room temperature. Immediately place the plate on dry ice, and add 1 mL 80% methanol/water (both HPLC grade) (pre-cooled in -80oC for at least 1hr).Remove the plate from -80oC freezer and put it on dry ice, scrape cells into extraction solvent. Transfer the whole cell extract to a new Eppendorf tube placed on ice. Centrifuge at 20 000 rcf for 10 min, 4oC.Transfer the supernatant into two tubes and dry with a speed vacuum at room temperature. The dried metabolites were stored at -80 freezer before analysis.","PROCESSING_STORAGE_CONDITIONS":"Described in summary","EXTRACT_STORAGE":"Described in summary"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"PFPP A gradient separation on a Restek Ultra PFPP column (150 x 2.1 mm, 3 μm; Restek, Bellefonte, PA) was used to separate the analytes. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 100% acetonitrile. The gradient began with 2 minutes at 0% B followed by a ramp to 25% B between 2 and 5 minutes, another ramp to 35% B between 5 and 11 minutes, a final increase from 35% to 95% B between 11 and 15 minutes, a hold at 95% B from 15 to 20 minutes, then a decrease to 0% B between 20 and 20.10 minutes, and a final hold at 0% B from 20.10 to 25 minutes. The flow rate to achieve separation was 0.25 ml/min. Ionization in the negative ESI mode occurred in the DUIS source with the following conditions: nebulizing gas flow of 2 L/min, heating gas flow of 5 L/min, interface temperature of 350°C, DL temperature of 250°C, heat block temperature of 400°C, and a drying gas flow of 15 L/min.","INSTRUMENT_NAME":"Shimadzu Nexera X2","COLUMN_NAME":"Restek Ultra PFPP (150 x 2.1mm,3um)","FLOW_GRADIENT":"The gradient began with 2 minutes at 0% B followed by a ramp to 25% B between 2 and 5 minutes, another ramp to 35% B between 5 and 11 minutes, a final increase from 35% to 95% B between 11 and 15 minutes, a hold at 95% B from 15 to 20 minutes, then a decrease to 0% B between 20 and 20.10 minutes, and a final hold at 0% B from 20.10 to 25 minutes.","FLOW_RATE":"0.25 ml/min","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile","CHROMATOGRAPHY_TYPE":"Other"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Shimadzu Nexera X2","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","MS_COMMENTS":"The analysis was performed on a Shimadzu Nexera UHPLC system coupled with a Shimadzu LCMS-8050 triple-quadrupole mass spectrometer (Kyoto, Japan). Two LC-MS/MS methods were employed to measure the targets.","ION_MODE":"NEGATIVE"},

"MS_METABOLITE_DATA":{
"Units":"Peak area ratio",

"Data":[{"Metabolite":"Citrate/Isocitrate","GSDMD-LPS-1":"7.0220","GSDMD-LPS-2":"8.4980","GSDMD-LPS-3":"10.9830","GSDMD-LPS-4":"8.2710","GSDMD-ATP-1":"5.3300","GSDMD-ATP-2":"8.3200","GSDMD-ATP-3":"12.9110","GSDMD-ATP-4":"6.4650","GSDMD-JX-1":"9.7820","GSDMD-JX-2":"11.2200","GSDMD-JX-3":"13.0720","GSDMD-JX-4":"12.5190","GSDMD-control-1":"2.7060","GSDMD-control-2":"5.4950","GSDMD-control-3":"5.0450","GSDMD-control-4":"8.5770","WT-LPS-1":"5.1240","WT-LPS-2":"4.8600","WT-LPS-3":"4.9040","WT-LPS-4":"5.7650","WT-ATP-1":"4.5580","WT-ATP-2":"8.9670","WT-ATP-3":"4.8240","WT-ATP-4":"3.2790","WT-JX-1":"10.7510","WT-JX-2":"4.7270","WT-JX-3":"3.9460","WT-JX-4":"3.1670","WT-control-1":"3.3330","WT-control-2":"4.5700","WT-control-3":"2.7730","WT-control-4":"3.8570"},{"Metabolite":"Fumaric Acid","GSDMD-LPS-1":"0.0110","GSDMD-LPS-2":"0.0100","GSDMD-LPS-3":"0.0150","GSDMD-LPS-4":"0.0170","GSDMD-ATP-1":"0.0150","GSDMD-ATP-2":"0.0180","GSDMD-ATP-3":"0.0300","GSDMD-ATP-4":"0.0140","GSDMD-JX-1":"0.0140","GSDMD-JX-2":"0.0170","GSDMD-JX-3":"0.0210","GSDMD-JX-4":"0.0200","GSDMD-control-1":"0.0030","GSDMD-control-2":"0.0050","GSDMD-control-3":"0.0030","GSDMD-control-4":"0.0050","WT-LPS-1":"0.0100","WT-LPS-2":"0.0060","WT-LPS-3":"0.0080","WT-LPS-4":"0.0100","WT-ATP-1":"0.0140","WT-ATP-2":"0.0130","WT-ATP-3":"0.0110","WT-ATP-4":"0.0160","WT-JX-1":"0.0140","WT-JX-2":"0.0090","WT-JX-3":"0.0100","WT-JX-4":"0.0070","WT-control-1":"0.0040","WT-control-2":"0.0020","WT-control-3":"0.0190","WT-control-4":"0.0050"},{"Metabolite":"Lactic Acid","GSDMD-LPS-1":"8.2300","GSDMD-LPS-2":"7.4980","GSDMD-LPS-3":"9.1980","GSDMD-LPS-4":"6.5430","GSDMD-ATP-1":"2.1950","GSDMD-ATP-2":"2.1560","GSDMD-ATP-3":"4.2970","GSDMD-ATP-4":"1.9870","GSDMD-JX-1":"2.6630","GSDMD-JX-2":"2.6320","GSDMD-JX-3":"3.2420","GSDMD-JX-4":"3.7440","GSDMD-control-1":"0.8720","GSDMD-control-2":"1.6990","GSDMD-control-3":"1.5780","GSDMD-control-4":"3.2270","WT-LPS-1":"3.3380","WT-LPS-2":"3.0750","WT-LPS-3":"4.4800","WT-LPS-4":"5.2680","WT-ATP-1":"2.4830","WT-ATP-2":"3.5150","WT-ATP-3":"3.5750","WT-ATP-4":"2.5310","WT-JX-1":"2.3490","WT-JX-2":"1.2450","WT-JX-3":"2.0360","WT-JX-4":"1.6400","WT-control-1":"0.7810","WT-control-2":"1.1450","WT-control-3":"0.9390","WT-control-4":"1.1040"},{"Metabolite":"Malic Acid","GSDMD-LPS-1":"0.9580","GSDMD-LPS-2":"1.0470","GSDMD-LPS-3":"1.4320","GSDMD-LPS-4":"1.0950","GSDMD-ATP-1":"0.5710","GSDMD-ATP-2":"0.9110","GSDMD-ATP-3":"1.5560","GSDMD-ATP-4":"0.8550","GSDMD-JX-1":"0.8230","GSDMD-JX-2":"0.7710","GSDMD-JX-3":"1.2290","GSDMD-JX-4":"1.3610","GSDMD-control-1":"0.2050","GSDMD-control-2":"0.3790","GSDMD-control-3":"0.4280","GSDMD-control-4":"0.7730","WT-LPS-1":"0.7220","WT-LPS-2":"0.6250","WT-LPS-3":"0.8200","WT-LPS-4":"1.0090","WT-ATP-1":"0.6230","WT-ATP-2":"1.4860","WT-ATP-3":"1.2030","WT-ATP-4":"1.0090","WT-JX-1":"1.1010","WT-JX-2":"0.6610","WT-JX-3":"0.9380","WT-JX-4":"0.7510","WT-control-1":"0.2650","WT-control-2":"0.3450","WT-control-3":"0.3960","WT-control-4":"0.4320"},{"Metabolite":"Succinic acid","GSDMD-LPS-1":"0.5130","GSDMD-LPS-2":"0.4950","GSDMD-LPS-3":"0.7190","GSDMD-LPS-4":"0.6160","GSDMD-ATP-1":"0.4470","GSDMD-ATP-2":"0.5350","GSDMD-ATP-3":"0.9480","GSDMD-ATP-4":"0.4630","GSDMD-JX-1":"0.5050","GSDMD-JX-2":"0.5140","GSDMD-JX-3":"0.6270","GSDMD-JX-4":"0.6480","GSDMD-control-1":"0.1080","GSDMD-control-2":"0.1980","GSDMD-control-3":"0.2310","GSDMD-control-4":"0.4830","WT-LPS-1":"0.5410","WT-LPS-2":"0.3790","WT-LPS-3":"0.5920","WT-LPS-4":"0.7180","WT-ATP-1":"0.5010","WT-ATP-2":"0.9300","WT-ATP-3":"0.8670","WT-ATP-4":"0.7570","WT-JX-1":"0.5620","WT-JX-2":"0.2880","WT-JX-3":"0.4570","WT-JX-4":"0.3810","WT-control-1":"0.1900","WT-control-2":"0.2220","WT-control-3":"0.3620","WT-control-4":"0.2990"}],

"Metabolites":[{"Metabolite":"Citrate/Isocitrate","pubchem_id":"1198","inchi_key":"","kegg_id":"C00311","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""},{"Metabolite":"Fumaric Acid","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""},{"Metabolite":"Lactic Acid","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""},{"Metabolite":"Malic Acid","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""},{"Metabolite":"Succinic acid","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"","ri":"","ri_type":"","moverz_quant":"","":""}]
}

}