#METABOLOMICS WORKBENCH Zhulab_20221116_105018 DATATRACK_ID:3579 STUDY_ID:ST002378 ANALYSIS_ID:AN003876 VERSION 1 CREATED_ON 12-15-2022 #PROJECT PR:PROJECT_TITLE Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation PR:PROJECT_TITLE during acute inflammation PR:PROJECT_TYPE Basic research PR:PROJECT_SUMMARY Activating macrophage NLRP3 inflammasome can promote excessive inflammation, PR:PROJECT_SUMMARY with severe cell and tissue damage and organ dysfunction. Here, we show that PR:PROJECT_SUMMARY pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) PR:PROJECT_SUMMARY significantly attenuates NLRP3 inflammasome activation in murine and human PR:PROJECT_SUMMARY macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta PR:PROJECT_SUMMARY secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic PR:PROJECT_SUMMARY reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, PR:PROJECT_SUMMARY preserves cristae ultrastructure, and attenuates mitochondrial ROS production. PR:PROJECT_SUMMARY The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is PR:PROJECT_SUMMARY independent of its canonical role as a pyruvate dehydrogenase regulator. We PR:PROJECT_SUMMARY suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 PR:PROJECT_SUMMARY inflammasome activation in acutely inflamed macrophages. PR:INSTITUTE Wake Forest School of Medicine PR:LAST_NAME Zhu PR:FIRST_NAME Xuewei PR:ADDRESS 575 Patterson Ave PR:EMAIL xwzhu@wakehealth.edu PR:PHONE 3367131445 PR:DOI http://dx.doi.org/10.21228/M8Q13W #STUDY ST:STUDY_TITLE Targeted metabolomics analysis of WT and GSDMDKO macrophages ST:STUDY_TYPE MS analysis ST:STUDY_SUMMARY Activating macrophage NLRP3 inflammasome can promote excessive inflammation, ST:STUDY_SUMMARY with severe cell and tissue damage and organ dysfunction. Here, we show that ST:STUDY_SUMMARY pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) ST:STUDY_SUMMARY significantly attenuates NLRP3 inflammasome activation in murine and human ST:STUDY_SUMMARY macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta ST:STUDY_SUMMARY secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic ST:STUDY_SUMMARY reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, ST:STUDY_SUMMARY preserves cristae ultrastructure, and attenuates mitochondrial ROS production. ST:STUDY_SUMMARY The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is ST:STUDY_SUMMARY independent of its canonical role as a pyruvate dehydrogenase regulator. We ST:STUDY_SUMMARY suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 ST:STUDY_SUMMARY inflammasome activation in acutely inflamed macrophages. ST:INSTITUTE Wake Forest School of Medicine ST:LAST_NAME Zhu ST:FIRST_NAME Xuewei ST:ADDRESS 575 Patterson Ave, Winston-Salem, NC 27101 ST:EMAIL xwzhu@wakehealth.edu ST:PHONE 3367131445 ST:SUBMIT_DATE 2022-11-16 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - GSDMD-LPS-1 Genotype:GSDMDKO | Treatment:LPS RAW_FILE_NAME=GSDMD-LPS-1.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-LPS-2 Genotype:GSDMDKO | Treatment:LPS RAW_FILE_NAME=GSDMD-LPS-2.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-LPS-3 Genotype:GSDMDKO | Treatment:LPS RAW_FILE_NAME=GSDMD-LPS-3.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-LPS-4 Genotype:GSDMDKO | Treatment:LPS RAW_FILE_NAME=GSDMD-LPS-4.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-ATP-1 Genotype:GSDMDKO | Treatment:LPS+ATP RAW_FILE_NAME=GSDMD-ATP-1.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-ATP-2 Genotype:GSDMDKO | Treatment:LPS+ATP RAW_FILE_NAME=GSDMD-ATP-2.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-ATP-3 Genotype:GSDMDKO | Treatment:LPS+ATP RAW_FILE_NAME=GSDMD-ATP-3.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-ATP-4 Genotype:GSDMDKO | Treatment:LPS+ATP RAW_FILE_NAME=GSDMD-ATP-4.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-JX-1 Genotype:GSDMDKO | Treatment:LPS+JX06+ATP RAW_FILE_NAME=GSDMD-JX-1.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-JX-2 Genotype:GSDMDKO | Treatment:LPS+JX06+ATP RAW_FILE_NAME=GSDMD-JX-2.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-JX-3 Genotype:GSDMDKO | Treatment:LPS+JX06+ATP RAW_FILE_NAME=GSDMD-JX-3.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-JX-4 Genotype:GSDMDKO | Treatment:LPS+JX06+ATP RAW_FILE_NAME=GSDMD-JX-4.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-control-1 Genotype:GSDMDKO | Treatment:no treatment RAW_FILE_NAME=GSDMD-control-1.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-control-2 Genotype:GSDMDKO | Treatment:no treatment RAW_FILE_NAME=GSDMD-control-2.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-control-3 Genotype:GSDMDKO | Treatment:no treatment RAW_FILE_NAME=GSDMD-control-3.mzml SUBJECT_SAMPLE_FACTORS - GSDMD-control-4 Genotype:GSDMDKO | Treatment:no treatment RAW_FILE_NAME=GSDMD-control-4.mzml SUBJECT_SAMPLE_FACTORS - WT-LPS-1 Genotype:WT | Treatment:LPS RAW_FILE_NAME=WT-LPS-1.mzml SUBJECT_SAMPLE_FACTORS - WT-LPS-2 Genotype:WT | Treatment:LPS RAW_FILE_NAME=WT-LPS-2.mzml SUBJECT_SAMPLE_FACTORS - WT-LPS-3 Genotype:WT | Treatment:LPS RAW_FILE_NAME=WT-LPS-3.mzml SUBJECT_SAMPLE_FACTORS - WT-LPS-4 Genotype:WT | Treatment:LPS RAW_FILE_NAME=WT-LPS-4.mzml SUBJECT_SAMPLE_FACTORS - WT-ATP-1 Genotype:WT | Treatment:LPS+ATP RAW_FILE_NAME=WT-ATP-1.mzml SUBJECT_SAMPLE_FACTORS - WT-ATP-2 Genotype:WT | Treatment:LPS+ATP RAW_FILE_NAME=WT-ATP-2.mzml SUBJECT_SAMPLE_FACTORS - WT-ATP-3 Genotype:WT | Treatment:LPS+ATP RAW_FILE_NAME=WT-ATP-3.mzml SUBJECT_SAMPLE_FACTORS - WT-ATP-4 Genotype:WT | Treatment:LPS+ATP RAW_FILE_NAME=WT-ATP-4.mzml SUBJECT_SAMPLE_FACTORS - WT-JX-1 Genotype:WT | Treatment:LPS+JX06+ATP RAW_FILE_NAME=WT-JX-1.mzml SUBJECT_SAMPLE_FACTORS - WT-JX-2 Genotype:WT | Treatment:LPS+JX06+ATP RAW_FILE_NAME=WT-JX-2.mzml SUBJECT_SAMPLE_FACTORS - WT-JX-3 Genotype:WT | Treatment:LPS+JX06+ATP RAW_FILE_NAME=WT-JX-3.mzml SUBJECT_SAMPLE_FACTORS - WT-JX-4 Genotype:WT | Treatment:LPS+JX06+ATP RAW_FILE_NAME=WT-JX-4.mzml SUBJECT_SAMPLE_FACTORS - WT-control-1 Genotype:WT | Treatment:no treatment RAW_FILE_NAME=WT-control-1.mzml SUBJECT_SAMPLE_FACTORS - WT-control-2 Genotype:WT | Treatment:no treatment RAW_FILE_NAME=WT-control-2.mzml SUBJECT_SAMPLE_FACTORS - WT-control-3 Genotype:WT | Treatment:no treatment RAW_FILE_NAME=WT-control-3.mzml SUBJECT_SAMPLE_FACTORS - WT-control-4 Genotype:WT | Treatment:no treatment RAW_FILE_NAME=WT-control-4.mzml #COLLECTION CO:COLLECTION_SUMMARY Macrophages were lysed, and polar metabolites were extracted using methanol and CO:COLLECTION_SUMMARY H2O (80:20; HPLC Grade; Sigma-Aldrich). Briefly, After treatment, immediately CO:COLLECTION_SUMMARY aspirate medium at room temperature. Immediately place the plate on dry ice, and CO:COLLECTION_SUMMARY add 1 mL 80% methanol/water (both HPLC grade) (pre-cooled in -80oC for at least CO:COLLECTION_SUMMARY 1hr).Remove the plate from -80oC freezer and put it on dry ice, scrape cells CO:COLLECTION_SUMMARY into extraction solvent. Transfer the whole cell extract to a new Eppendorf tube CO:COLLECTION_SUMMARY placed on ice. Centrifuge at 20 000 rcf for 10 min, 4oC.Transfer the supernatant CO:COLLECTION_SUMMARY into two tubes and dry with a speed vacuum at room temperature. CO:SAMPLE_TYPE Macrophages CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY WT and GSDMD KO bone marrow derived macrophages were first primed with 300 ng/ml TR:TREATMENT_SUMMARY LPS (E. coli 0111; B4, Sigma-Aldrich) before stimulated with or without 5 mM ATP TR:TREATMENT_SUMMARY (Sigma-Aldrich) for 30 min in the presence or absence of 10 uM JX06. Here, we TR:TREATMENT_SUMMARY show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase TR:TREATMENT_SUMMARY (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and TR:TREATMENT_SUMMARY human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta TR:TREATMENT_SUMMARY secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic TR:TREATMENT_SUMMARY reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, TR:TREATMENT_SUMMARY preserves cristae ultrastructure, and attenuates mitochondrial ROS production. TR:TREATMENT_SUMMARY The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is TR:TREATMENT_SUMMARY independent of its canonical role as a pyruvate dehydrogenase regulator. We TR:TREATMENT_SUMMARY suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 TR:TREATMENT_SUMMARY inflammasome activation in acutely inflamed macrophages. TR:TREATMENT In vitro culture TR:TREATMENT_COMPOUND LPS, ATP, JX06 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Macrophages were lysed, and polar metabolites were extracted using methanol and SP:SAMPLEPREP_SUMMARY H2O (80:20; HPLC Grade; Sigma-Aldrich). Briefly, After treatment, immediately SP:SAMPLEPREP_SUMMARY aspirate medium at room temperature. Immediately place the plate on dry ice, and SP:SAMPLEPREP_SUMMARY add 1 mL 80% methanol/water (both HPLC grade) (pre-cooled in -80oC for at least SP:SAMPLEPREP_SUMMARY 1hr).Remove the plate from -80oC freezer and put it on dry ice, scrape cells SP:SAMPLEPREP_SUMMARY into extraction solvent. Transfer the whole cell extract to a new Eppendorf tube SP:SAMPLEPREP_SUMMARY placed on ice. Centrifuge at 20 000 rcf for 10 min, 4oC.Transfer the supernatant SP:SAMPLEPREP_SUMMARY into two tubes and dry with a speed vacuum at room temperature. The dried SP:SAMPLEPREP_SUMMARY metabolites were stored at -80 freezer before analysis. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY PFPP A gradient separation on a Restek Ultra PFPP column (150 x 2.1 mm, 3 μm; CH:CHROMATOGRAPHY_SUMMARY Restek, Bellefonte, PA) was used to separate the analytes. Mobile phase A CH:CHROMATOGRAPHY_SUMMARY consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid in 100% acetonitrile. The gradient began with 2 minutes at 0% B CH:CHROMATOGRAPHY_SUMMARY followed by a ramp to 25% B between 2 and 5 minutes, another ramp to 35% B CH:CHROMATOGRAPHY_SUMMARY between 5 and 11 minutes, a final increase from 35% to 95% B between 11 and 15 CH:CHROMATOGRAPHY_SUMMARY minutes, a hold at 95% B from 15 to 20 minutes, then a decrease to 0% B between CH:CHROMATOGRAPHY_SUMMARY 20 and 20.10 minutes, and a final hold at 0% B from 20.10 to 25 minutes. The CH:CHROMATOGRAPHY_SUMMARY flow rate to achieve separation was 0.25 ml/min. Ionization in the negative ESI CH:CHROMATOGRAPHY_SUMMARY mode occurred in the DUIS source with the following conditions: nebulizing gas CH:CHROMATOGRAPHY_SUMMARY flow of 2 L/min, heating gas flow of 5 L/min, interface temperature of 350°C, CH:CHROMATOGRAPHY_SUMMARY DL temperature of 250°C, heat block temperature of 400°C, and a drying gas CH:CHROMATOGRAPHY_SUMMARY flow of 15 L/min. CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Restek Ultra PFPP (150 x 2.1mm,3um) CH:FLOW_GRADIENT The gradient began with 2 minutes at 0% B followed by a ramp to 25% B between 2 CH:FLOW_GRADIENT and 5 minutes, another ramp to 35% B between 5 and 11 minutes, a final increase CH:FLOW_GRADIENT from 35% to 95% B between 11 and 15 minutes, a hold at 95% B from 15 to 20 CH:FLOW_GRADIENT minutes, then a decrease to 0% B between 20 and 20.10 minutes, and a final hold CH:FLOW_GRADIENT at 0% B from 20.10 to 25 minutes. CH:FLOW_RATE 0.25 ml/min CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile CH:CHROMATOGRAPHY_TYPE Other #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu Nexera X2 MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS The analysis was performed on a Shimadzu Nexera UHPLC system coupled with a MS:MS_COMMENTS Shimadzu LCMS-8050 triple-quadrupole mass spectrometer (Kyoto, Japan). Two MS:MS_COMMENTS LC-MS/MS methods were employed to measure the targets. MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area ratio MS_METABOLITE_DATA_START Samples GSDMD-LPS-1 GSDMD-LPS-2 GSDMD-LPS-3 GSDMD-LPS-4 GSDMD-ATP-1 GSDMD-ATP-2 GSDMD-ATP-3 GSDMD-ATP-4 GSDMD-JX-1 GSDMD-JX-2 GSDMD-JX-3 GSDMD-JX-4 GSDMD-control-1 GSDMD-control-2 GSDMD-control-3 GSDMD-control-4 WT-LPS-1 WT-LPS-2 WT-LPS-3 WT-LPS-4 WT-ATP-1 WT-ATP-2 WT-ATP-3 WT-ATP-4 WT-JX-1 WT-JX-2 WT-JX-3 WT-JX-4 WT-control-1 WT-control-2 WT-control-3 WT-control-4 Factors Genotype:GSDMDKO | Treatment:LPS Genotype:GSDMDKO | Treatment:LPS Genotype:GSDMDKO | Treatment:LPS Genotype:GSDMDKO | Treatment:LPS Genotype:GSDMDKO | Treatment:LPS+ATP Genotype:GSDMDKO | Treatment:LPS+ATP Genotype:GSDMDKO | Treatment:LPS+ATP Genotype:GSDMDKO | Treatment:LPS+ATP Genotype:GSDMDKO | Treatment:LPS+JX06+ATP Genotype:GSDMDKO | Treatment:LPS+JX06+ATP Genotype:GSDMDKO | Treatment:LPS+JX06+ATP Genotype:GSDMDKO | Treatment:LPS+JX06+ATP Genotype:GSDMDKO | Treatment:no treatment Genotype:GSDMDKO | Treatment:no treatment Genotype:GSDMDKO | Treatment:no treatment Genotype:GSDMDKO | Treatment:no treatment Genotype:WT | Treatment:LPS Genotype:WT | Treatment:LPS Genotype:WT | Treatment:LPS Genotype:WT | Treatment:LPS Genotype:WT | Treatment:LPS+ATP Genotype:WT | Treatment:LPS+ATP Genotype:WT | Treatment:LPS+ATP Genotype:WT | Treatment:LPS+ATP Genotype:WT | Treatment:LPS+JX06+ATP Genotype:WT | Treatment:LPS+JX06+ATP Genotype:WT | Treatment:LPS+JX06+ATP Genotype:WT | Treatment:LPS+JX06+ATP Genotype:WT | Treatment:no treatment Genotype:WT | Treatment:no treatment Genotype:WT | Treatment:no treatment Genotype:WT | Treatment:no treatment Citrate/Isocitrate 7.0220 8.4980 10.9830 8.2710 5.3300 8.3200 12.9110 6.4650 9.7820 11.2200 13.0720 12.5190 2.7060 5.4950 5.0450 8.5770 5.1240 4.8600 4.9040 5.7650 4.5580 8.9670 4.8240 3.2790 10.7510 4.7270 3.9460 3.1670 3.3330 4.5700 2.7730 3.8570 Fumaric Acid 0.0110 0.0100 0.0150 0.0170 0.0150 0.0180 0.0300 0.0140 0.0140 0.0170 0.0210 0.0200 0.0030 0.0050 0.0030 0.0050 0.0100 0.0060 0.0080 0.0100 0.0140 0.0130 0.0110 0.0160 0.0140 0.0090 0.0100 0.0070 0.0040 0.0020 0.0190 0.0050 Lactic Acid 8.2300 7.4980 9.1980 6.5430 2.1950 2.1560 4.2970 1.9870 2.6630 2.6320 3.2420 3.7440 0.8720 1.6990 1.5780 3.2270 3.3380 3.0750 4.4800 5.2680 2.4830 3.5150 3.5750 2.5310 2.3490 1.2450 2.0360 1.6400 0.7810 1.1450 0.9390 1.1040 Malic Acid 0.9580 1.0470 1.4320 1.0950 0.5710 0.9110 1.5560 0.8550 0.8230 0.7710 1.2290 1.3610 0.2050 0.3790 0.4280 0.7730 0.7220 0.6250 0.8200 1.0090 0.6230 1.4860 1.2030 1.0090 1.1010 0.6610 0.9380 0.7510 0.2650 0.3450 0.3960 0.4320 Succinic acid 0.5130 0.4950 0.7190 0.6160 0.4470 0.5350 0.9480 0.4630 0.5050 0.5140 0.6270 0.6480 0.1080 0.1980 0.2310 0.4830 0.5410 0.3790 0.5920 0.7180 0.5010 0.9300 0.8670 0.7570 0.5620 0.2880 0.4570 0.3810 0.1900 0.2220 0.3620 0.2990 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant Citrate/Isocitrate 1198 C00311 Fumaric Acid Lactic Acid Malic Acid Succinic acid METABOLITES_END #END