#METABOLOMICS WORKBENCH kmaus13_20230116_075919 DATATRACK_ID:3691 STUDY_ID:ST002445 ANALYSIS_ID:AN003983 PROJECT_ID:PR001576 VERSION 1 CREATED_ON January 17, 2023, 12:14 pm #PROJECT PR:PROJECT_TITLE Mouse Neutrophil NTEM eicosanoids WT vs KI vs KO PR:PROJECT_SUMMARY Uncontrolled inflammation is linked to poor outcomes in sepsis and wound PR:PROJECT_SUMMARY healing, which are multi-phased physiological responses. Eicosanoids, a class of PR:PROJECT_SUMMARY bioactive lipids, play a major regulatory role in these physiologies. In this PR:PROJECT_SUMMARY study, the ablation of the ceramide-1-phosphate (C1P) interaction site in the PR:PROJECT_SUMMARY eicosanoid biosynthetic enzyme, group IVA cytosolic phospholipase A2, in mice PR:PROJECT_SUMMARY (cPLA2a-KI mice) resulted in enhanced and sustained neutrophil infiltration into PR:PROJECT_SUMMARY both wounds and the peritoneum during the inflammatory phase of wound healing PR:PROJECT_SUMMARY and sepsis. Enhanced neutrophil infiltration (i.e., neutrophilia) was associated PR:PROJECT_SUMMARY with significant improvements in wound healing and the survival of mice to PR:PROJECT_SUMMARY sepsis. As neutrophilia at the site of injury or infection normally associates PR:PROJECT_SUMMARY with a poor outcome, our laboratory investigated this "Neutrophil Conundrum" by PR:PROJECT_SUMMARY characterizing the cPLA2a-KI neutrophils, which showed enhanced N2-subtype PR:PROJECT_SUMMARY markers, trans-endothelial migration, phagocytosis and VEGF with a concomitant PR:PROJECT_SUMMARY decrease in TNFa, neutrophil extracellular trap production, N1-subtype markers, PR:PROJECT_SUMMARY and endothelial cell damage versus wild-type neutrophils. This N2 polarization PR:PROJECT_SUMMARY of cPLA2a-KI neutrophils was due to an induction of the 5-HETE/5-oxo-ETE PR:PROJECT_SUMMARY biosynthetic pathway and activation of the OXER1 receptor. Unbiased proteomics PR:PROJECT_SUMMARY identified perturbations in the pentose phosphate pathway (PPP) specific to PR:PROJECT_SUMMARY OXER1 signaling in the cPLA2a-KI neutrophils, and modulation of the PPP PR:PROJECT_SUMMARY recapitulated specific aspects of the N2 phenotype. Thus, C1P via cPLA2a PR:PROJECT_SUMMARY negatively regulates 5-oxo-ETE biosynthesis, which controls neutrophil PR:PROJECT_SUMMARY polarization via the PPP PR:INSTITUTE University of South Florida PR:DEPARTMENT CAS PR:LABORATORY Chalfant PR:LAST_NAME Maus PR:FIRST_NAME Kenneth PR:ADDRESS 4202 E Fowler Ave, Tampa, FL, 33620, USA PR:EMAIL kmaus@usf.edu PR:PHONE 8139283137 #STUDY ST:STUDY_TITLE The interaction of ceramide-1-phosphate with group IVA cytosolic phospholipase ST:STUDY_TITLE A2 modulates neutrophil polarization during inflammatory responses ST:STUDY_SUMMARY Bone marrow-derived mouse neutrophils from wildtype, cPLA2alpha-knockin ST:STUDY_SUMMARY (interaction site on cPLA2alpha for its substrate C1P was ablated), and ST:STUDY_SUMMARY cPLA2alpha-knockout (cPLA2alpha gene mutated) mice were exposed to 4 hours of ST:STUDY_SUMMARY trans-endothelial migration and resulting eicosanoids were analyzed (eg, PGE2, ST:STUDY_SUMMARY PGD2, 5-HETE, and 5-oxo-ETE). ST:INSTITUTE University of South Florida ST:LAST_NAME Maus ST:FIRST_NAME Kenneth ST:ADDRESS 4202 E Fowler Ave, CMMB - NES 107 - Chalfant Lab ST:EMAIL kmaus@usf.edu ST:PHONE 8139283137 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN balbc, blk6 background SU:AGE_OR_AGE_RANGE 10-14 weeks SU:GENDER Male and female SU:ANIMAL_HOUSING USF College of MEdicine SU:ANIMAL_LIGHT_CYCLE Normal SU:ANIMAL_FEED Ad libdum SU:ANIMAL_WATER Ad libdum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Kenny 1 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 2 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 3 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 4 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 5 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 6 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 7 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 8 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 9 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 10 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 11 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 12 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 13 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny 14 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny and Anika Eicosanoids_reinject.WIFF SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 1 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 2 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 3 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 4 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 5 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 6 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 7 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 8 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 9 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 10 Genotype:Wild-type | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 11 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 12 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 13 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 14 Genotype:cPLA2a-KI | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 15 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 16 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 17 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff SUBJECT_SAMPLE_FACTORS - Kenny Eicosanoids 18 Genotype:cPLA2a-KO | Treatment:NTEM RAW_FILE_NAME=Kenny Eicosanoids.wiff #COLLECTION CO:COLLECTION_SUMMARY HUVECs were seeded on the upper wells Corning® Transwell® polycarbonate CO:COLLECTION_SUMMARY membrane inserts with 3.0 µm pores (Sigma-Aldrich CLS3415) inserted into a CO:COLLECTION_SUMMARY 24-well tissue culture plate at a density of 1.6 x 106 cells per ml in 250 µl CO:COLLECTION_SUMMARY volume. Inflammatory response was induced by adding TNFα (Cayman #32020, 20 CO:COLLECTION_SUMMARY ng/ml) 16-18 hours before addition of PNs. PNs were then isolated from mouse CO:COLLECTION_SUMMARY bone marrow and added to the upper well while HUVEC media containing CO:COLLECTION_SUMMARY lipopolysaccharides (LPS) from E. coli (Millipore Sigma #L3023, 1 µg/ml) was CO:COLLECTION_SUMMARY added to the bottom well as a chemoattractant. After time indicated (e.g., 4 CO:COLLECTION_SUMMARY hours), media and cells from the upper and lower wells were extracted, counted, CO:COLLECTION_SUMMARY combined, centrifuged, and washed in preparation for UHPLC-MS/MS. CO:SAMPLE_TYPE Bone marrow #TREATMENT TR:TREATMENT_SUMMARY Eicosanoids and/or inhibitors (Cayman Chemical) were applied at the following TR:TREATMENT_SUMMARY concentrations: 1.0 nM 5-HETE (#34210), 1.0 nM 5-oxo-ETE (#34250), 7.5 nM MK886 TR:TREATMENT_SUMMARY (#21753), 25 µM Gue1654 (#29686), 100 nM physcion (Santa Cruz Biotech TR:TREATMENT_SUMMARY SC-205805). Cells were allowed to equilibrate with inhibitors for 30 minutes TR:TREATMENT_SUMMARY before experimentation via NTEM and subsequent collection and processing for TR:TREATMENT_SUMMARY UHPLC-MS/MS. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Media collection: Add proper volumes to achieve final concentrations of 10% MeOH SP:SAMPLEPREP_SUMMARY and 0.5% glacial acetic acid. Then add 20 μL eicosanoid internal standard. SP:SAMPLEPREP_SUMMARY Example: For 4 mL media add 400 μL of 100% MeOH and 20 μL of glacial acetic SP:SAMPLEPREP_SUMMARY acid. • Example, for 24 samples, prep for 26: o 2.6 mL 100% MeOH (100 μL x SP:SAMPLEPREP_SUMMARY 26) o 130 μL glacial acetic acid (5 μL x 26) o 520 μL eicosanoid IS (20 μL x SP:SAMPLEPREP_SUMMARY 26) Columns: Set the manometer on the vacuum chamber to 5 mmHg. Precondition SP:SAMPLEPREP_SUMMARY columns: Elute 2 mL MeOH, stop, then elute 2 mL H2O. Load sample and tighten the SP:SAMPLEPREP_SUMMARY caps. Wash: Add 2 mL of 5% MeOH to the sample vial. Vortex and apply to the SP:SAMPLEPREP_SUMMARY column under vacuum. Allow to run dry for 30 seconds. Stop vacuum, remove SP:SAMPLEPREP_SUMMARY manifold & lay on side. Change out glass vials and elute: Apply 2 mL Isopropyl SP:SAMPLEPREP_SUMMARY alcohol to column and equilibrate for 1 minute. Elute under vacuum and run dry SP:SAMPLEPREP_SUMMARY for 30 seconds. Concentration: The solvent is removed by speed vac for !5 hours. SP:SAMPLEPREP_SUMMARY Eicosanoids are re-dissolved in 100 μL of 50% EtOH in HPLC water. Centrifuge @ SP:SAMPLEPREP_SUMMARY 4000 RPM for 10 minutes to pellet. Transfer supernatant into glass 0.1 mL mini SP:SAMPLEPREP_SUMMARY conicals and load into white slots in HPLC tray. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexara X2 LC-30AD CH:COLUMN_NAME MilliporeSigma Supelco Ascentis Express C18 (150 x 4.6 mm, 2.7 um) CH:SOLVENT_A 20% acetonitrile/80% water; 0.02% formic acid CH:SOLVENT_B 20% acetonitrile/80% isopropanol; 0.02% formic acid CH:FLOW_GRADIENT The column was equilibrated with 100% Solvent A for 5 min and then 10 µl of CH:FLOW_GRADIENT sample was injected. 100% Solvent A was used for the first two minutes of CH:FLOW_GRADIENT elution. Solvent B was increased in a linear gradient to 25% Solvent B at 3 min, CH:FLOW_GRADIENT to 30% at 6 min., to 55% at 6.1 min, to 70% at 10 min, and to 100% at 10.10 min. CH:FLOW_GRADIENT 100% Solvent B was held constant until 13.0 min, where it was decreased to 0% CH:FLOW_GRADIENT Solvent B and 100% Solvent A from 13.0 min to 13.1 min. From 13.1 min to 14.0 CH:FLOW_GRADIENT min. Solvent A was held constant at 100% CH:FLOW_RATE 0.5 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Eicosanoids were analyzed via mass spec using an AB Sciex Triple Quad 5500 Mass MS:MS_COMMENTS Spectrometer as previously described by Mitra et al., 2007 (FEBS Lett 581, MS:MS_COMMENTS 581–793). Q1 and Q3 were set to detect distinctive precursor and product ion MS:MS_COMMENTS pairs. Ions were fragmented in Q2 using N2 gas for collisionally induced MS:MS_COMMENTS dissociation. Analysis used multiple-reaction monitoring in negative-ion mode. MS:MS_COMMENTS Eicosanoids were monitored using precursor → product MRM pairs. The mass MS:MS_COMMENTS spectrometer parameters as previously described by Chalfant et al., 2001 and MS:MS_COMMENTS Wijesinghe et al., 2013 were: Curtain Gas: 20; CAD: Medium; Ion Spray Voltage: MS:MS_COMMENTS -4500V; Temperature: 300°C; Gas 1: 40; Gas 2: 60; Declustering Potential, MS:MS_COMMENTS Collision Energy, and Cell Exit Potential vary per transition #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmol/mL MS_METABOLITE_DATA_START Samples Kenny 1 Kenny 2 Kenny 3 Kenny 4 Kenny 5 Kenny 6 Kenny 7 Kenny 8 Kenny 9 Kenny 10 Kenny 11 Kenny 12 Kenny 13 Kenny 14 Kenny Eicosanoids 1 Kenny Eicosanoids 2 Kenny Eicosanoids 3 Kenny Eicosanoids 4 Kenny Eicosanoids 5 Kenny Eicosanoids 6 Kenny Eicosanoids 7 Kenny Eicosanoids 8 Kenny Eicosanoids 9 Kenny Eicosanoids 10 Kenny Eicosanoids 11 Kenny Eicosanoids 12 Kenny Eicosanoids 13 Kenny Eicosanoids 14 Kenny Eicosanoids 15 Kenny Eicosanoids 16 Kenny Eicosanoids 17 Kenny Eicosanoids 18 Factors Genotype:Wild-type | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:Wild-type | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KI | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM Genotype:cPLA2a-KO | Treatment:NTEM PGE2 7.58E+03 5.27E+03 4.67E+03 5.29E+03 6.18E+03 4.18E+03 4.70E+05 4.60E+05 4.50E+05 6.78E+05 4.96E+05 3.98E+05 4.65E+05 3.77E+05 4.03E+05 1.60E+05 1.37E+05 2.17E+05 4.40E+05 3.87E+05 1.02E+05 2.49E+05 6.01E+05 7.00E+05 5.22E+05 8.84E+03 1.40E+05 1.14E+05 1.14E+06 1.12E+06 1.19E+06 1.22E+06 PGD2 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 1.38E+05 1.19E+05 1.58E+05 2.15E+05 1.47E+05 1.23E+05 1.24E+05 1.25E+05 2.83E+04 9.96E+03 5.12E+03 7.80E+03 2.85E+04 1.69E+04 8.09E+03 1.77E+04 4.45E+04 3.72E+04 2.36E+04 8.53E+02 1.03E+04 5.76E+03 6.22E+04 5.77E+04 6.58E+04 8.15E+04 5 HETE 1.40E+05 8.07E+04 1.25E+05 9.24E+04 1.01E+05 1.27E+05 4.79E+04 3.89E+04 4.36E+04 5.87E+04 4.59E+04 4.86E+04 6.80E+04 6.50E+04 9.14E+04 4.69E+04 3.49E+04 6.45E+04 1.01E+05 7.77E+04 5.13E+03 1.61E+04 3.90E+04 5.31E+04 2.63E+04 6.40E+02 5.94E+03 4.87E+03 4.37E+04 3.53E+04 3.51E+04 4.01E+04 5-OxoETE 8.29E+03 5.62E+03 7.66E+03 5.19E+03 5.51E+03 1.51E+04 9.68E+03 1.62E+04 7.14E+03 1.05E+04 7.12E+03 8.10E+03 1.54E+04 1.17E+04 8.29E+03 3.13E+03 3.06E+03 3.73E+03 7.31E+03 5.47E+03 0.00E+00 1.30E+03 2.47E+03 2.69E+03 1.92E+03 1.95E+02 6.28E+02 3.75E+02 4.60E+03 3.65E+03 2.11E+03 1.93E+03 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID PGE2 5280360 C00584 PGD2 448457 C00696 5 HETE 5280733 C04805 5-OxoETE 5283159 C14732 METABOLITES_END #END