#METABOLOMICS WORKBENCH Zhulab_20221116_105018 DATATRACK_ID:3579 STUDY_ID:ST002378 ANALYSIS_ID:AN003875 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 500 μL of cell extract and 20 μL of MES (Thermo Fisher Scientific, Waltham, CH:CHROMATOGRAPHY_SUMMARY MA, USA) internal standard solution (10 ng/μL) were combined and mixed CH:CHROMATOGRAPHY_SUMMARY thoroughly through vortexing. The mixture was then dried under vacuum and CH:CHROMATOGRAPHY_SUMMARY reconstituted for analysis in 100 μL of ultrapure water (Optima, Thermo Fisher CH:CHROMATOGRAPHY_SUMMARY Scientific, Waltham, MA, USA). The analysis was performed on a Shimadzu Nexera CH:CHROMATOGRAPHY_SUMMARY UHPLC system coupled with a Shimadzu LCMS-8050 triple-quadrupole mass CH:CHROMATOGRAPHY_SUMMARY spectrometer (Kyoto, Japan). Two LC-MS/MS methods were employed to measure the CH:CHROMATOGRAPHY_SUMMARY targets. Ion-Pairing Separation was performed at 0.3 ml/min on a Zorbax Eclipse CH:CHROMATOGRAPHY_SUMMARY Plus C18 column (1.8 μm, 2.1 x 100 mm; Agilent, Santa Clara, CA USA). Mobile CH:CHROMATOGRAPHY_SUMMARY phase A consisted of ultrapure water (Optima, Thermo Fisher Scientific, Waltham, CH:CHROMATOGRAPHY_SUMMARY MA, USA) with 10 mM ammonium acetate (J.T. Baker, Thermo Fisher Scientific, CH:CHROMATOGRAPHY_SUMMARY Waltham, MA, USA) and 10 mM tributylamine (Acros Organics, Thermo Fisher CH:CHROMATOGRAPHY_SUMMARY Scientific, Fair Lawn NJ, USA) and mobile phase B consisted of methanol (Optima, CH:CHROMATOGRAPHY_SUMMARY Thermo Fisher Scientific, Waltham, MA, USA). The separation used the following CH:CHROMATOGRAPHY_SUMMARY gradient profile: 2 minutes at 0% B, a ramp to 25% B at 8 minutes, another ramp CH:CHROMATOGRAPHY_SUMMARY to 98%B at 12 minutes, a 3 minute hold until 15 minutes, and then a drop back to CH:CHROMATOGRAPHY_SUMMARY 0% B at 15.1 minutes and allowed to equilibrate there until 25 minutes. All CH:CHROMATOGRAPHY_SUMMARY analytes were monitored in negative mode. CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) CH:FLOW_GRADIENT The separation used the following gradient profile: 2 minutes at 0% B, a ramp to CH:FLOW_GRADIENT 25% B at 8 minutes, another ramp to 98%B at 12 minutes, a 3 minute hold until 15 CH:FLOW_GRADIENT minutes, and then a drop back to 0% B at 15.1 minutes and allowed to equilibrate CH:FLOW_GRADIENT there until 25 minutes. CH:SOLVENT_A 100% water; 10 mM ammonium acetate; 10 mM tributylamine CH:SOLVENT_B 100% methanol CH:CHROMATOGRAPHY_TYPE Ion pair #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 2_3-bis-Phosphoglycerate 0.0170 0.0250 0.0190 0.0180 0.7740 1.4330 1.2100 1.1290 0.8470 0.6020 1.1140 1.3360 0.0190 0.0200 0.0430 0.0370 0.0250 0.0590 0.0230 0.0290 0.7440 0.8030 0.5900 0.3870 0.7860 1.1090 0.8520 0.8160 0.0140 0.0360 0.0230 0.0070 3-Phosphoglycerate/2-Phosphoglycerate 0.0590 0.0660 0.0800 0.0500 1.3520 1.6860 1.4040 1.8720 1.4860 0.5650 2.1930 2.0280 0.1080 0.0820 0.0590 0.0620 0.0440 0.1160 0.0480 0.0620 0.9140 0.7550 1.1070 0.5370 1.2930 1.3640 1.7130 1.5530 0.0720 0.0940 0.0760 0.0630 Fructose-1_6-bis-phosphate 0.1410 0.1710 0.1350 0.0690 1.7660 2.5070 2.1990 2.4570 1.8780 1.3190 3.3510 2.6440 0.1730 0.1400 0.1800 0.0580 0.1420 0.2140 0.0920 0.1960 1.1360 0.9340 1.3920 0.9660 1.7020 2.2370 2.4240 1.7630 0.1300 0.1320 0.0330 0.0380 Glucose-1-phosphate 0.0260 0.0360 0.0460 0.0690 0.0870 0.1120 0.1480 0.1220 0.1100 0.0720 0.1450 0.1810 0.0170 0.0110 0.0230 0.0090 0.0090 0.0160 0.0370 0.0240 0.2200 0.2350 0.2370 0.1430 0.1280 0.1290 0.1650 0.1540 0.0090 0.0120 0.0160 0.0160 Glucose-6-phosphate/Fructose-6-phosphate 0.0470 0.0270 0.0760 0.1570 0.1870 0.2120 0.2220 0.2500 0.2160 0.1360 0.2890 0.3520 0.0120 0.0360 0.0280 0.0210 0.0100 0.0390 0.0580 0.0440 0.5410 0.4890 0.5700 0.3200 0.2780 0.2670 0.3100 0.3140 0.0130 0.0150 0.0090 0.0280 Glyceraldehyde-3-phosphate/Dihydroxyacetonephosphate 0.0170 0.0280 0.0270 0.1020 0.6500 0.3270 0.3300 0.3300 0.8130 0.4700 0.8800 1.1540 0.0040 0.0110 0.0090 0.0200 0.0320 0.0340 0.0090 0.0260 1.0560 0.3540 0.5300 0.1780 0.5000 0.3730 0.3980 0.5000 0.0050 0.0030 0.0070 0.0060 Glycerol-3-phosphate 0.1230 0.1310 0.1590 0.1690 0.0840 0.0950 0.1530 0.1090 0.0960 0.0500 0.1350 0.1610 0.0360 0.0740 0.0580 0.0770 0.0860 0.0700 0.1190 0.1170 0.1220 0.1410 0.1300 0.0600 0.1070 0.0920 0.1210 0.1210 0.0210 0.0410 0.0290 0.0370 Phosphoenolpyruvate 0.0200 0.0310 0.0100 0.0080 1.0290 1.4210 1.3000 1.3740 1.1740 0.7530 1.6010 1.6700 0.0140 0.0060 0.0110 0.0050 0.0140 0.0360 0.0110 0.0260 0.7450 0.8790 0.8520 0.5170 1.0730 1.2860 1.3660 1.3690 0.0100 0.0150 0.0070 0.0050 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 2,3-bis-Phosphoglycerate 3-Phosphoglycerate/2-Phosphoglycerate 439278 C00631 Fructose-1,6-bis-phosphate Glucose-1-phosphate Glucose-6-phosphate/Fructose-6-phosphate 440641 C05345 Glyceraldehyde-3-phosphate/Dihydroxyacetonephosphate 668 C00111 Glycerol-3-phosphate Phosphoenolpyruvate METABOLITES_END #END