Summary of Study ST002371

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001524. The data can be accessed directly via it's Project DOI: 10.21228/M8Q13W This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002371
Study TitleHigh-resolution metabolomics analysis of NLRP3 inflammasome activated macrophages
Study TypeMS analysis
Study SummaryActivating 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 NameZhu
First NameXuewei
Address575 Pattern Ave.
Emailxwzhu@wakehealth.edu
Phone3367131445
Submit Date2022-11-15
Num Groups3
Total Subjects12
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailGC-MS
Release Date2022-12-15
Release Version1
Xuewei Zhu Xuewei Zhu
https://dx.doi.org/10.21228/M8Q13W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001524
Project DOI:doi: 10.21228/M8Q13W
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

Subject:

Subject ID:SU002460
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA237321LA-2LPS+ATP
SA237322LA-4LPS+ATP
SA237323LA-1LPS+ATP
SA237324LA-3LPS+ATP
SA237325LJA-3LPS+JX06+ATP
SA237326LJA-4LPS+JX06+ATP
SA237327LJA-2LPS+JX06+ATP
SA237328LJA-1LPS+JX06+ATP
SA237329Control-2No treatment
SA237330Control-1No treatment
SA237331Control-3No treatment
SA237332Control-4No treatment
Showing results 1 to 12 of 12

Collection:

Collection ID:CO002453
Collection Summary:Mouse bone marrow was cultured in low glucose DMEM supplemented with 30% L929 cell-conditioned medium, 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin for 6-7 days until the cells reached confluence. BMDMs were then reseeded in culture dishes overnight in RPMI-1640 medium containing 1% Nutridoma-SP medium (Sigma-Aldrich). LPS-primed BMDMs were stimulated with 5 mM ATP in the presence or absence of 10 uM JX06 for 45 min. Unstimulated macrophages were used as control.
Sample Type:Macrophages
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002472
Treatment Summary:Macrophages were treated with 300 ng/ml LPS for 3 h. LPS-primed BMDMs were stimulated with 5 mM ATP in the presence or absence of 10 uM JX06 for 45 min. Unstimulated macrophages were used as control.
Treatment Compound:LPS, ATP, JX06

Sample Preparation:

Sampleprep ID:SP002466
Sampleprep Summary:Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich) (Liu et al., 2015). Extracts were dried in a vacuum concentrator at room temperature and stored at -80 freezer.
Processing Storage Conditions:Described in summary
Extraction Method:methanol and H2O
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003866
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Dionex Ultimate 3000
Column Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode UNSPECIFIED
Units peak area

Chromatography:

Chromatography ID:CH002864
Chromatography Summary:The HPLC (Ultimate 3000 UHPLC) is coupled to QE-MS (Thermo Scientific) for metabolite separation and detection. An Xbridge amide column (100 × 2.1 mm i.d., 3.5 μm; Waters) is employed for compound separation at room temperature. The mobile phase A is water containing 5 mM ammonium acetate (pH 6.8), and the mobile phase B is acetonitrile. The linear gradient used is as follows: 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B. The flow rate was 0.15 mL/min from 0 to 10 min and 15 to 20 min and 0.3 mL/min from 10.5 to 14.5 min. The QE-MS is equipped with a HESI probe with related parameters set as below: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.0 kV for the positive mode and 2.5 kV for the negative mode; capillary temperature, 320 °C; S-lens, 55; scan range (m/z): 70 to 900 for pos mode (1.31 to 12.5 min) and neg mode (1.31 to 6.6 min) and 100 to 1000 for neg mode (6.61 to 12.5 min); resolution: 70000; automated gain control (AGC), 3 × 106 ions. Customized mass calibration was performed before data acquisition.
Methods Filename:The_HPLC_methods.docx
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters XBridge Amide (100 x 4.6mm,3.5um)
Flow Gradient:The linear gradient used is as follows: 0 min, 85% B; 1.5 min, 85% B, 5.5 min, 35% B; 10 min, 35% B, 10.5 min, 35% B, 14.5 min, 35% B, 15 min, 85% B, and 20 min, 85% B.
Flow Rate:0.15 mL/min from 0 to 10 min and 15 to 20 min and 0.3 mL/min from 10.5 to 14.5 min.
Solvent A:100% water; 5 mM ammonium acetate, pH 6.8
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS003607
Analysis ID:AN003866
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:LC-MS peak extraction and integration were performed using commercial available software Sieve 2.2 (Thermo Scientific). The peak area was used to represent the relative abundance of each metabolite in different samples.
Ion Mode:UNSPECIFIED
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