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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST002445 |
| Study Title | The interaction of ceramide-1-phosphate with group IVA cytosolic phospholipase A2 modulates neutrophil polarization during inflammatory responses |
| Study Summary | Bone 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 Name | Maus |
| First Name | Kenneth |
| Address | 4202 E Fowler Ave, CMMB - NES 107 - Chalfant Lab |
| kmaus@usf.edu | |
| Phone | 8139283137 |
| Submit Date | 2023-01-16 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | wiff |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-02-05 |
| Release Version | 1 |
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 |
| Species Group: | Mammals |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Treatment |
|---|---|---|---|
| SA244625 | Kenny Eicosanoids 4 | cPLA2a-KI | NTEM |
| SA244626 | Kenny Eicosanoids 12 | cPLA2a-KI | NTEM |
| SA244627 | Kenny Eicosanoids 14 | cPLA2a-KI | NTEM |
| SA244628 | Kenny Eicosanoids 13 | cPLA2a-KI | NTEM |
| SA244629 | Kenny Eicosanoids 11 | cPLA2a-KI | NTEM |
| SA244630 | Kenny Eicosanoids 3 | cPLA2a-KI | NTEM |
| SA244631 | Kenny 7 | cPLA2a-KI | NTEM |
| SA244632 | Kenny Eicosanoids 2 | cPLA2a-KI | NTEM |
| SA244633 | Kenny 8 | cPLA2a-KI | NTEM |
| SA244634 | Kenny 5 | cPLA2a-KI | NTEM |
| SA244635 | Kenny 6 | cPLA2a-KI | NTEM |
| SA244636 | Kenny Eicosanoids 16 | cPLA2a-KO | NTEM |
| SA244637 | Kenny Eicosanoids 18 | cPLA2a-KO | NTEM |
| SA244638 | Kenny Eicosanoids 17 | cPLA2a-KO | NTEM |
| SA244639 | Kenny Eicosanoids 15 | cPLA2a-KO | NTEM |
| SA244640 | Kenny 9 | cPLA2a-KO | NTEM |
| SA244641 | Kenny Eicosanoids 6 | cPLA2a-KO | NTEM |
| SA244642 | Kenny Eicosanoids 5 | cPLA2a-KO | NTEM |
| SA244643 | Kenny Eicosanoids 7 | cPLA2a-KO | NTEM |
| SA244644 | Kenny 12 | cPLA2a-KO | NTEM |
| SA244645 | Kenny 11 | cPLA2a-KO | NTEM |
| SA244646 | Kenny 10 | cPLA2a-KO | NTEM |
| SA244615 | Kenny 1 | Wild-type | NTEM |
| SA244616 | Kenny 14 | Wild-type | NTEM |
| SA244617 | Kenny Eicosanoids 10 | Wild-type | NTEM |
| SA244618 | Kenny Eicosanoids 9 | Wild-type | NTEM |
| SA244619 | Kenny Eicosanoids 1 | Wild-type | NTEM |
| SA244620 | Kenny 13 | Wild-type | NTEM |
| SA244621 | Kenny 2 | Wild-type | NTEM |
| SA244622 | Kenny 3 | Wild-type | NTEM |
| SA244623 | Kenny 4 | Wild-type | NTEM |
| SA244624 | Kenny Eicosanoids 8 | Wild-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. |
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 |
Analysis:
| Analysis ID: | AN003983 |
| Analysis Type: | MS |
| Chromatography ID: | CH002945 |
| Num Factors: | 3 |
| Num Metabolites: | 4 |
| Units: | pmol/mL |
