Summary of Study ST002445

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 PR001576. The data can be accessed directly via it's Project DOI: 10.21228/M80709 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 IDST002445
Study TitleThe interaction of ceramide-1-phosphate with group IVA cytosolic phospholipase A2 modulates neutrophil polarization during inflammatory responses
Study SummaryBone marrow-derived mouse neutrophils from wildtype, cPLA2alpha-knockin (interaction site on cPLA2alpha for its substrate C1P was ablated), and cPLA2alpha-knockout (cPLA2alpha gene mutated) mice were exposed to 4 hours of trans-endothelial migration and resulting eicosanoids were analyzed (eg, PGE2, PGD2, 5-HETE, and 5-oxo-ETE).
Institute
University of South Florida
Last NameMaus
First NameKenneth
Address4202 E Fowler Ave, CMMB - NES 107 - Chalfant Lab
Emailkmaus@usf.edu
Phone8139283137
Submit Date2023-01-16
Raw Data AvailableYes
Raw Data File Type(s)wiff
Analysis Type DetailLC-MS
Release Date2023-02-05
Release Version1
Kenneth Maus Kenneth Maus
https://dx.doi.org/10.21228/M80709
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001576
Project DOI:doi: 10.21228/M80709
Project Title:Mouse Neutrophil NTEM eicosanoids WT vs KI vs KO
Project Summary:Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, which are multi-phased physiological responses. Eicosanoids, a class of bioactive lipids, play a major regulatory role in these physiologies. In this study, the ablation of the ceramide-1-phosphate (C1P) interaction site in the eicosanoid biosynthetic enzyme, group IVA cytosolic phospholipase A2, in mice (cPLA2a-KI mice) resulted in enhanced and sustained neutrophil infiltration into both wounds and the peritoneum during the inflammatory phase of wound healing and sepsis. Enhanced neutrophil infiltration (i.e., neutrophilia) was associated with significant improvements in wound healing and the survival of mice to sepsis. As neutrophilia at the site of injury or infection normally associates with a poor outcome, our laboratory investigated this Neutrophil Conundrum by characterizing the cPLA2a-KI neutrophils, which showed enhanced N2-subtype markers, trans-endothelial migration, phagocytosis and VEGF with a concomitant decrease in TNFa, neutrophil extracellular trap production, N1-subtype markers, and endothelial cell damage versus wild-type neutrophils. This N2 polarization of cPLA2a-KI neutrophils was due to an induction of the 5-HETE/5-oxo-ETE biosynthetic pathway and activation of the OXER1 receptor. Unbiased proteomics identified perturbations in the pentose phosphate pathway (PPP) specific to OXER1 signaling in the cPLA2a-KI neutrophils, and modulation of the PPP recapitulated specific aspects of the N2 phenotype. Thus, C1P via cPLA2a negatively regulates 5-oxo-ETE biosynthesis, which controls neutrophil polarization via the PPP
Institute:University of South Florida
Department:CAS
Laboratory:Chalfant
Last Name:Maus
First Name:Kenneth
Address:4202 E Fowler Ave, Tampa, FL, 33620, USA
Email:kmaus@usf.edu
Phone:8139283137

Subject:

Subject ID:SU002534
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:balbc, blk6 background
Age Or Age Range:10-14 weeks
Gender:Male and female
Animal Housing:USF College of MEdicine
Animal Light Cycle:Normal
Animal Feed:Ad libdum
Animal Water:Ad libdum

Factors:

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

mb_sample_id local_sample_id Genotype Treatment
SA244625Kenny Eicosanoids 4cPLA2a-KI NTEM
SA244626Kenny Eicosanoids 12cPLA2a-KI NTEM
SA244627Kenny Eicosanoids 14cPLA2a-KI NTEM
SA244628Kenny Eicosanoids 13cPLA2a-KI NTEM
SA244629Kenny Eicosanoids 11cPLA2a-KI NTEM
SA244630Kenny Eicosanoids 3cPLA2a-KI NTEM
SA244631Kenny 7cPLA2a-KI NTEM
SA244632Kenny Eicosanoids 2cPLA2a-KI NTEM
SA244633Kenny 8cPLA2a-KI NTEM
SA244634Kenny 5cPLA2a-KI NTEM
SA244635Kenny 6cPLA2a-KI NTEM
SA244636Kenny Eicosanoids 16cPLA2a-KO NTEM
SA244637Kenny Eicosanoids 18cPLA2a-KO NTEM
SA244638Kenny Eicosanoids 17cPLA2a-KO NTEM
SA244639Kenny Eicosanoids 15cPLA2a-KO NTEM
SA244640Kenny 9cPLA2a-KO NTEM
SA244641Kenny Eicosanoids 6cPLA2a-KO NTEM
SA244642Kenny Eicosanoids 5cPLA2a-KO NTEM
SA244643Kenny Eicosanoids 7cPLA2a-KO NTEM
SA244644Kenny 12cPLA2a-KO NTEM
SA244645Kenny 11cPLA2a-KO NTEM
SA244646Kenny 10cPLA2a-KO NTEM
SA244615Kenny 1Wild-type NTEM
SA244616Kenny 14Wild-type NTEM
SA244617Kenny Eicosanoids 10Wild-type NTEM
SA244618Kenny Eicosanoids 9Wild-type NTEM
SA244619Kenny Eicosanoids 1Wild-type NTEM
SA244620Kenny 13Wild-type NTEM
SA244621Kenny 2Wild-type NTEM
SA244622Kenny 3Wild-type NTEM
SA244623Kenny 4Wild-type NTEM
SA244624Kenny Eicosanoids 8Wild-type NTEM
Showing results 1 to 32 of 32

Collection:

Collection ID:CO002527
Collection Summary:HUVECs were seeded on the upper wells Corning® Transwell® polycarbonate membrane inserts with 3.0 µm pores (Sigma-Aldrich CLS3415) inserted into a 24-well tissue culture plate at a density of 1.6 x 106 cells per ml in 250 µl volume. Inflammatory response was induced by adding TNFα (Cayman #32020, 20 ng/ml) 16-18 hours before addition of PNs. PNs were then isolated from mouse bone marrow and added to the upper well while HUVEC media containing lipopolysaccharides (LPS) from E. coli (Millipore Sigma #L3023, 1 µg/ml) was added to the bottom well as a chemoattractant. After time indicated (e.g., 4 hours), media and cells from the upper and lower wells were extracted, counted, combined, centrifuged, and washed in preparation for UHPLC-MS/MS.
Sample Type:Bone marrow

Treatment:

Treatment ID:TR002546
Treatment Summary:Eicosanoids and/or inhibitors (Cayman Chemical) were applied at the following concentrations: 1.0 nM 5-HETE (#34210), 1.0 nM 5-oxo-ETE (#34250), 7.5 nM MK886 (#21753), 25 µM Gue1654 (#29686), 100 nM physcion (Santa Cruz Biotech SC-205805). Cells were allowed to equilibrate with inhibitors for 30 minutes before experimentation via NTEM and subsequent collection and processing for UHPLC-MS/MS.

Sample Preparation:

Sampleprep ID:SP002540
Sampleprep Summary:Media collection: Add proper volumes to achieve final concentrations of 10% MeOH and 0.5% glacial acetic acid. Then add 20 μL eicosanoid internal standard. Example: For 4 mL media add 400 μL of 100% MeOH and 20 μL of glacial acetic acid. • Example, for 24 samples, prep for 26: o 2.6 mL 100% MeOH (100 μL x 26) o 130 μL glacial acetic acid (5 μL x 26) o 520 μL eicosanoid IS (20 μL x 26) Columns: Set the manometer on the vacuum chamber to 5 mmHg. Precondition columns: Elute 2 mL MeOH, stop, then elute 2 mL H2O. Load sample and tighten the caps. Wash: Add 2 mL of 5% MeOH to the sample vial. Vortex and apply to the column under vacuum. Allow to run dry for 30 seconds. Stop vacuum, remove manifold & lay on side. Change out glass vials and elute: Apply 2 mL Isopropyl alcohol to column and equilibrate for 1 minute. Elute under vacuum and run dry for 30 seconds. Concentration: The solvent is removed by speed vac for !5 hours. Eicosanoids are re-dissolved in 100 μL of 50% EtOH in HPLC water. Centrifuge @ 4000 RPM for 10 minutes to pellet. Transfer supernatant into glass 0.1 mL mini conicals and load into white slots in HPLC tray.

Combined analysis:

Analysis ID AN003983
Analysis type MS
Chromatography type Reversed phase
Chromatography system Shimadzu Nexara X2 LC-30AD
Column MilliporeSigma Supelco Ascentis Express C18 (150 x 4.6 mm, 2.7 um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name ABI Sciex 5500 QTrap
Ion Mode NEGATIVE
Units pmol/mL

Chromatography:

Chromatography ID:CH002945
Instrument Name:Shimadzu Nexara X2 LC-30AD
Column Name:MilliporeSigma Supelco Ascentis Express C18 (150 x 4.6 mm, 2.7 um)
Column Temperature:40
Flow Gradient:The column was equilibrated with 100% Solvent A for 5 min and then 10 µl of sample was injected. 100% Solvent A was used for the first two minutes of elution. Solvent B was increased in a linear gradient to 25% Solvent B at 3 min, to 30% at 6 min., to 55% at 6.1 min, to 70% at 10 min, and to 100% at 10.10 min. 100% Solvent B was held constant until 13.0 min, where it was decreased to 0% Solvent B and 100% Solvent A from 13.0 min to 13.1 min. From 13.1 min to 14.0 min. Solvent A was held constant at 100%
Flow Rate:0.5 ml/min
Solvent A:20% acetonitrile/80% water; 0.02% formic acid
Solvent B:20% acetonitrile/80% isopropanol; 0.02% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003717
Analysis ID:AN003983
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Eicosanoids were analyzed via mass spec using an AB Sciex Triple Quad 5500 Mass Spectrometer as previously described by Mitra et al., 2007 (FEBS Lett 581, 581–793). Q1 and Q3 were set to detect distinctive precursor and product ion pairs. Ions were fragmented in Q2 using N2 gas for collisionally induced dissociation. Analysis used multiple-reaction monitoring in negative-ion mode. Eicosanoids were monitored using precursor → product MRM pairs. The mass spectrometer parameters as previously described by Chalfant et al., 2001 and Wijesinghe et al., 2013 were: Curtain Gas: 20; CAD: Medium; Ion Spray Voltage: -4500V; Temperature: 300°C; Gas 1: 40; Gas 2: 60; Declustering Potential, Collision Energy, and Cell Exit Potential vary per transition
Ion Mode:NEGATIVE
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