#METABOLOMICS WORKBENCH juliehaines_20211122_120944 DATATRACK_ID:2939 STUDY_ID:ST001998 ANALYSIS_ID:AN003260 PROJECT_ID:000000 VERSION 1 CREATED_ON November 22, 2021, 12:25 pm #PROJECT PR:PROJECT_TITLE Polyamine import and accumulation causes immunomodulation in macrophages PR:PROJECT_TITLE engulfing apoptotic cells PR:PROJECT_SUMMARY Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue PR:PROJECT_SUMMARY homeostasis and drives anti-inflammatory programming in engulfing macrophages. PR:PROJECT_SUMMARY Here, we assess metabolites in naïve and inflammatory macrophages following PR:PROJECT_SUMMARY engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to PR:PROJECT_SUMMARY unique increases in the arginine-derived polyamines, spermidine and spermine, in PR:PROJECT_SUMMARY vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does PR:PROJECT_SUMMARY not arise from retention of apoptotic cell metabolites or de novo synthesis, but PR:PROJECT_SUMMARY from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. PR:PROJECT_SUMMARY Blocking polyamine import prevents efferocytosis from suppressing macrophage PR:PROJECT_SUMMARY IL-1beta or IL-6. This identifies efferocytosis as a trigger for polyamine PR:PROJECT_SUMMARY import and accumulation, and imported polyamines as mediators of PR:PROJECT_SUMMARY efferocytosis-induced immune reprogramming. PR:INSTITUTE University of Colorado Denver PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE Polyamine import and accumulation causes immunomodulation in macrophages ST:STUDY_TITLE engulfing apoptotic cells (Part 3) ST:STUDY_SUMMARY Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue ST:STUDY_SUMMARY homeostasis and drives anti-inflammatory programming in engulfing macrophages. ST:STUDY_SUMMARY Here, we assess metabolites in naïve and inflammatory macrophages following ST:STUDY_SUMMARY engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to ST:STUDY_SUMMARY unique increases in the arginine-derived polyamines, spermidine and spermine, in ST:STUDY_SUMMARY vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does ST:STUDY_SUMMARY not arise from retention of apoptotic cell metabolites or de novo synthesis, but ST:STUDY_SUMMARY from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. ST:STUDY_SUMMARY Blocking polyamine import prevents efferocytosis from suppressing macrophage ST:STUDY_SUMMARY IL-1beta or IL-6. This identifies efferocytosis as a trigger for polyamine ST:STUDY_SUMMARY import and accumulation, and imported polyamines as mediators of ST:STUDY_SUMMARY efferocytosis-induced immune reprogramming. ST:INSTITUTE University of Colorado Denver ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - unexp1 category:unexposed macrophages RAW_FILE_NAME=14 SUBJECT_SAMPLE_FACTORS - unexp2 category:unexposed macrophages RAW_FILE_NAME=17 SUBJECT_SAMPLE_FACTORS - unexp3 category:unexposed macrophages RAW_FILE_NAME=20 SUBJECT_SAMPLE_FACTORS - 1 - category:non-engulfing RAW_FILE_NAME=15 SUBJECT_SAMPLE_FACTORS - 2 - category:non-engulfing RAW_FILE_NAME=18 SUBJECT_SAMPLE_FACTORS - 3 - category:non-engulfing RAW_FILE_NAME=21 SUBJECT_SAMPLE_FACTORS - 1 + category:engulfing RAW_FILE_NAME=16 SUBJECT_SAMPLE_FACTORS - 2 + category:engulfing RAW_FILE_NAME=19 SUBJECT_SAMPLE_FACTORS - 3 + category:engulfing RAW_FILE_NAME=22 #COLLECTION CO:COLLECTION_SUMMARY LPS-primed murine peritoneal macrophages were fed fluorescently-tagged apoptotic CO:COLLECTION_SUMMARY Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were CO:COLLECTION_SUMMARY left to degrade targets for a further 3 hours. Macrophages were then collected CO:COLLECTION_SUMMARY and sorted into non-engulfing or engulfing populations, alongside control CO:COLLECTION_SUMMARY unexposed macrophages. Sorted cells were pelleted at 400xg and dry pellets were CO:COLLECTION_SUMMARY snap frozen and stored at -80C until metabolite extraction. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY LPS-primed murine peritoneal macrophages were fed fluorescently-tagged apoptotic TR:TREATMENT_SUMMARY Jurkat cells for 1h. Unengulfed targets were washed off and macrophages were TR:TREATMENT_SUMMARY left to degrade targets for a further 3 hours. Macrophages were then collected TR:TREATMENT_SUMMARY and sorted into non-engulfing or engulfing populations, alongside control TR:TREATMENT_SUMMARY unexposed macrophages. Sorted cells were pelleted at 400xg and dry pellets were TR:TREATMENT_SUMMARY snap frozen and stored at -80C until metabolite extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To process cells for assessment of intracellular metabolites, cells were SP:SAMPLEPREP_SUMMARY pelleted at 400xg in tubes coated with 0.06% BSA. Supernatant was aspirated and SP:SAMPLEPREP_SUMMARY discarded; residual liquid was carefully wicked away from the pellet with a SP:SAMPLEPREP_SUMMARY kimwipe. Dry pellets were immediately snap frozen and stored at -80C until SP:SAMPLEPREP_SUMMARY processing. To process culture supernatants for assessment of metabolites, SP:SAMPLEPREP_SUMMARY supernatant was centrifuged at 400xg to pellet any cells. Cell-free supernatant SP:SAMPLEPREP_SUMMARY was then transferred to a fresh tube, snap frozen, and stored at -80C until SP:SAMPLEPREP_SUMMARY processing. Ultra-high pressure liquid chromatography-mass spectrometry SP:SAMPLEPREP_SUMMARY (UHPLC-MS) was performed by the University of Colorado School of Medicine SP:SAMPLEPREP_SUMMARY Metabolomics Core. Metabolites from frozen cell pellets were extracted at 2e6 SP:SAMPLEPREP_SUMMARY cells/mL in ice cold 5:3:2 MeOH:acetonitrile:water (v/v/v). Media was thawed on SP:SAMPLEPREP_SUMMARY ice and a 10 L aliquot treated with 240 L of the same extraction solution. SP:SAMPLEPREP_SUMMARY Extractions were carried out using vigorous vortexing for 30 min at 4C. SP:SAMPLEPREP_SUMMARY Supernatants were clarified by centrifugation (10 min, 18,000 g, 4C) and SP:SAMPLEPREP_SUMMARY analyzed using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass SP:SAMPLEPREP_SUMMARY spectrometer. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Isocratic flow of 250 uL/min of 100% A (95% water, 5% acetonitrile, 10 mM CH:CHROMATOGRAPHY_SUMMARY ammonium acetate). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) CH:FLOW_GRADIENT isocratic CH:FLOW_RATE 250 ul/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Global metabolomics analyses were performed using a 3 min isocratic run in MS:MS_COMMENTS positive and negative ion modes (separate runs) as described previously (Nemkov MS:MS_COMMENTS et al., 2015, Nemkov et al., 2017); stable isotope tracing samples were analyzed MS:MS_COMMENTS using a 5 min C18 gradient in positive and negative ion modes (separate runs) as MS:MS_COMMENTS described (Nemkov et al., 2019, Gehrke et al., 2019). For all analyses, the MS MS:MS_COMMENTS scanned in MS1 mode across the m/z range of 65 to 950. Peaks were annotated (in MS:MS_COMMENTS conjunction with the KEGG database), integrated, and quality control performed MS:MS_COMMENTS using Maven (Princeton University) as described. Stable isotope tracing results MS:MS_COMMENTS were isotopically corrected for the natural abundance of 13C1, 13C2, 15N1, and MS:MS_COMMENTS 15N2 (Nemkov et al., 2017). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples unexp1 unexp2 unexp3 1 - 2 - 3 - 1 + 2 + 3 + Factors category:unexposed macrophages category:unexposed macrophages category:unexposed macrophages category:non-engulfing category:non-engulfing category:non-engulfing category:engulfing category:engulfing category:engulfing ADP 9.21E+03 8.93E+03 1.10E+04 1.50E+04 1.13E+04 1.13E+04 8.34E+03 1.09E+04 1.09E+04 Urate 1.31E+05 9.30E+04 1.16E+05 1.06E+05 1.15E+05 1.40E+05 2.19E+05 3.77E+05 2.28E+05 3',5'-Cyclic IMP 1.87E+04 2.98E+03 3.64E+04 2.42E+05 0.00E+00 5.03E+04 3.69E+05 0.00E+00 1.57E+04 Phosphate 7.74E+07 7.11E+07 7.07E+07 9.18E+07 7.59E+07 8.05E+07 7.49E+07 6.39E+07 6.14E+07 Diphosphate 9.41E+06 6.75E+06 7.42E+06 9.35E+06 1.04E+07 7.42E+06 5.89E+06 7.52E+06 5.11E+06 D-Glucose 3.20E+06 3.43E+06 3.12E+06 4.64E+06 1.32E+06 3.88E+06 5.99E+06 1.62E+06 3.72E+06 D-Glucose 6-phosphate 4.16E+05 3.19E+05 3.54E+05 5.80E+05 3.33E+05 4.96E+05 5.73E+05 5.99E+05 6.14E+05 Pyruvate 2.15E+05 1.88E+05 2.82E+05 3.42E+05 1.37E+05 2.69E+05 3.57E+05 2.43E+05 3.31E+05 Lactate 8.94E+05 9.93E+05 8.81E+05 1.84E+06 1.44E+06 1.10E+06 1.47E+06 1.84E+06 1.06E+06 D-Ribose 5.17E+04 4.98E+04 4.93E+04 7.07E+04 4.55E+04 5.54E+04 1.14E+05 4.38E+04 4.77E+04 Citrate 2.17E+06 1.49E+06 1.39E+06 2.86E+06 1.44E+06 2.28E+06 4.40E+06 2.19E+06 2.52E+06 2-Oxoglutarate 9.56E+04 8.81E+04 8.04E+04 1.57E+05 8.86E+04 8.62E+04 1.21E+05 1.41E+05 8.52E+04 Malate 4.99E+05 5.55E+05 1.98E+05 4.30E+05 2.52E+05 8.26E+05 6.21E+05 3.36E+05 4.18E+05 Oxaloacetate 1.37E+06 1.17E+06 1.44E+06 1.65E+06 1.43E+06 1.29E+06 1.30E+06 1.66E+06 1.19E+06 Itaconate 7.36E+05 8.01E+05 3.66E+04 8.99E+02 4.17E+03 7.60E+05 3.02E+05 3.84E+04 0.00E+00 2-Hydroxyglutarate/Citramalate 2.15E+05 1.98E+05 2.52E+05 2.58E+05 2.28E+05 3.47E+05 2.36E+05 2.90E+05 3.33E+05 alpha-D-Ribose 1-phosphate 7.97E+04 4.00E+04 8.59E+04 1.24E+05 5.47E+04 8.44E+04 1.32E+05 1.08E+05 1.68E+05 Ascorbate 1.94E+05 1.62E+05 1.89E+05 4.07E+05 7.57E+04 2.39E+05 4.81E+05 1.21E+05 2.30E+05 N-Acetylneuraminate 1.66E+04 2.37E+03 1.57E+04 1.81E+04 1.39E+04 2.62E+04 1.45E+04 1.08E+04 3.43E+04 alpha-D-Glucosamine 1-phosphate 9.03E+02 4.33E+03 8.72E+03 1.69E+04 1.61E+04 2.26E+03 6.85E+03 1.61E+04 1.77E+04 5-Hydroxyindoleacetate 0.00E+00 7.99E+02 9.92E+02 1.44E+03 9.43E+02 1.79E+03 2.40E+03 4.02E+03 2.45E+03 N-formyl kynurenine 6.39E+04 5.72E+04 7.48E+04 6.01E+04 5.25E+04 6.32E+04 7.84E+04 1.26E+05 1.54E+05 2-Oxoadipate 1.19E+05 1.48E+05 1.59E+05 2.13E+05 5.29E+04 1.78E+05 2.46E+05 8.78E+04 2.00E+05 Glycerol 3-phosphate 3.32E+04 1.53E+04 3.51E+04 5.33E+04 8.56E+03 5.03E+04 5.03E+04 4.23E+04 5.96E+04 Ethanolamine phosphate 6.02E+05 6.31E+05 6.51E+05 8.26E+05 5.15E+05 9.53E+05 7.00E+05 6.90E+05 9.39E+05 2-Methyleneglutarate 1.77E+05 1.89E+05 1.82E+05 2.75E+05 9.44E+04 2.33E+05 3.20E+05 1.36E+05 2.44E+05 6-Lactoyl-5-6-7-8-tetrahydropterin 1.79E+05 2.10E+05 2.19E+05 1.94E+05 1.44E+05 2.35E+05 2.20E+05 3.87E+05 3.69E+05 D-glucono-1,5-lactone 3.88E+05 4.32E+05 4.84E+05 6.17E+05 1.67E+05 4.63E+05 8.32E+05 2.45E+05 5.57E+05 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. ADP C00008 426.0205 2.08 Urate C00366 167.0202 1.18 3',5'-Cyclic IMP C00943 329.0289 1.24 Phosphate C00009 96.9677 1.19 Diphosphate C00013 176.9342 1.11 D-Glucose C00031 179.0549 1.20 D-Glucose 6-phosphate C00092 259.0209 1.10 Pyruvate C00022 87.0069 1.14 Lactate C01432 89.0225 1.25 D-Ribose C00121 149.0444 1.15 Citrate C00158 191.0189 1.15 2-Oxoglutarate C00026 145.0147 1.23 Malate C00149 133.0129 1.11 Oxaloacetate C00036 130.9999 1.18 Itaconate C00490 129.0177 1.45 2-Hydroxyglutarate/Citramalate C02630 147.0284 1.09 alpha-D-Ribose 1-phosphate C00620 229.0107 1.16 Ascorbate C00072 175.0250 1.22 N-Acetylneuraminate C00270 308.0974 1.11 alpha-D-Glucosamine 1-phosphate C06156 258.0366 1.11 5-Hydroxyindoleacetate C05635 190.0512 2.11 N-formyl kynurenine C02700 235.0746 1.19 2-Oxoadipate C00322 159.0285 1.17 Glycerol 3-phosphate C00093 171.0050 1.12 Ethanolamine phosphate C00346 140.0100 1.16 2-Methyleneglutarate C02930 143.0331 1.21 6-Lactoyl-5-6-7-8-tetrahydropterin C04244 238.0936 1.19 D-glucono-1,5-lactone C00198 177.0393 1.21 METABOLITES_END #END