#METABOLOMICS WORKBENCH juliehaines_20211122_122645 DATATRACK_ID:2940 STUDY_ID:ST001999 ANALYSIS_ID:AN003261 PROJECT_ID:000000 VERSION 1 CREATED_ON November 22, 2021, 12:33 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 4) 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 - ctrl 1 Category:unexposed macrophages RAW_FILE_NAME=7+ SUBJECT_SAMPLE_FACTORS - ctrl 2 Category:unexposed macrophages RAW_FILE_NAME=8+ SUBJECT_SAMPLE_FACTORS - ctrl 3 Category:unexposed macrophages RAW_FILE_NAME=9+ SUBJECT_SAMPLE_FACTORS - 1 -AC 8h Category:non-engulfing RAW_FILE_NAME=10+ SUBJECT_SAMPLE_FACTORS - 2 -AC 8h Category:non-engulfing RAW_FILE_NAME=11+ SUBJECT_SAMPLE_FACTORS - 3 -AC 8h Category:non-engulfing RAW_FILE_NAME=12+ SUBJECT_SAMPLE_FACTORS - 1 +AC 8h Category:engulfing RAW_FILE_NAME=13+ SUBJECT_SAMPLE_FACTORS - 2 +AC 8h Category:engulfing RAW_FILE_NAME=14+ SUBJECT_SAMPLE_FACTORS - 3 +AC 8h Category:engulfing RAW_FILE_NAME=15+ #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 7 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 7 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 at 250 uL/min using 95% phase A (0.1% formic acid in water) and CH:CHROMATOGRAPHY_SUMMARY 5% phase B (0.1% formic acid in acetonitrile). 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 POSITIVE 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 ctrl 1 ctrl 2 ctrl 3 1 -AC 8h 2 -AC 8h 3 -AC 8h 1 +AC 8h 2 +AC 8h 3 +AC 8h 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 L-alanine 6.70E+05 6.41E+05 6.96E+05 6.47E+05 7.43E+05 7.44E+05 6.68E+05 7.25E+05 6.57E+05 L-arginine 3.03E+05 2.99E+05 4.56E+05 1.70E+05 1.77E+05 1.49E+05 1.97E+06 1.36E+06 2.74E+06 L-asparagine 1.94E+04 3.90E+04 2.19E+04 0.00E+00 1.56E+04 1.68E+04 1.41E+05 8.35E+04 9.89E+04 L-aspartate 1.00E+05 8.02E+04 9.67E+04 1.53E+04 2.82E+04 2.65E+04 2.36E+05 1.52E+05 1.52E+05 L-cysteine 1.38E+05 1.39E+05 9.36E+04 1.18E+04 4.57E+04 1.33E+04 4.54E+05 4.20E+05 3.08E+05 L-glutamate 8.71E+05 7.34E+05 6.42E+05 2.17E+05 3.23E+05 2.45E+05 1.48E+06 9.97E+05 9.94E+05 L-glutamine 3.45E+05 3.62E+05 4.89E+05 1.72E+05 1.63E+05 1.23E+05 1.43E+06 1.07E+06 1.12E+06 L-histidine 7.33E+04 9.43E+04 1.33E+05 5.97E+04 6.12E+04 4.95E+04 4.49E+05 2.71E+05 3.39E+05 L-leucine 6.89E+05 9.60E+05 1.37E+06 5.49E+05 5.18E+05 6.71E+05 3.37E+06 2.68E+06 2.58E+06 L-lysine 1.86E+05 2.34E+05 4.05E+05 1.90E+05 1.95E+05 1.99E+05 3.11E+06 2.18E+06 2.52E+06 L-methionine 2.53E+04 2.46E+04 8.23E+03 0.00E+00 0.00E+00 0.00E+00 1.37E+05 8.78E+04 7.67E+04 L-phenylalanine 1.46E+05 1.91E+05 4.47E+05 2.67E+05 1.79E+05 2.45E+05 9.89E+05 7.37E+05 6.06E+05 L-proline 1.53E+06 2.81E+06 2.04E+06 1.47E+06 1.46E+06 1.70E+06 6.14E+06 4.90E+06 4.06E+06 L-serine 3.62E+04 6.12E+04 5.43E+04 2.54E+04 2.38E+04 1.23E+04 1.64E+05 8.43E+04 1.10E+05 L-threonine 1.03E+05 1.04E+05 9.32E+04 3.81E+04 5.73E+04 1.76E+04 4.88E+05 4.28E+05 3.41E+05 L-tryptophan 4.92E+05 5.83E+05 6.33E+05 7.15E+05 4.52E+05 7.64E+05 4.58E+06 3.86E+06 3.46E+06 L-tyrosine 2.05E+05 2.08E+05 2.61E+05 1.32E+05 1.84E+05 1.99E+05 4.75E+05 4.63E+05 3.60E+05 L-valine 2.94E+06 3.56E+06 3.03E+06 2.27E+06 2.61E+06 2.53E+06 4.00E+06 3.73E+06 2.74E+06 AMP 1.62E+05 1.90E+05 1.07E+05 5.40E+04 8.75E+04 6.60E+04 3.45E+05 1.98E+05 2.17E+05 Adenosine 3.45E+05 6.07E+05 3.83E+05 1.39E+05 1.51E+05 1.23E+05 3.15E+05 2.38E+05 7.77E+04 Cytosine 4.75E+04 1.18E+05 4.03E+04 1.96E+05 2.61E+05 3.34E+05 5.44E+06 5.53E+06 3.21E+06 IMP 1.13E+05 1.13E+05 5.93E+04 1.72E+04 2.06E+04 1.37E+04 1.12E+05 3.54E+04 5.01E+04 Hypoxanthine 3.12E+05 6.46E+05 2.76E+05 3.30E+05 3.12E+05 3.44E+05 3.39E+06 2.57E+06 1.40E+06 Urate 3.62E+04 3.72E+04 2.15E+04 9.78E+03 9.58E+03 1.55E+04 6.90E+04 3.91E+04 3.51E+04 Nicotinamide 3.18E+06 8.07E+06 3.38E+06 1.77E+06 2.50E+06 2.41E+06 7.33E+06 5.96E+06 1.86E+06 Thymine 5.87E+04 7.70E+04 6.32E+04 6.24E+04 6.03E+04 7.24E+04 1.30E+05 1.23E+05 1.56E+05 Glutathione 5.49E+05 6.56E+05 4.33E+05 3.57E+05 2.73E+05 2.02E+05 1.20E+06 9.13E+05 3.41E+05 S-Adenosyl-L-methionine 2.07E+04 4.78E+04 3.14E+04 0.00E+00 2.55E+04 2.65E+04 1.61E+05 9.08E+04 5.93E+04 Ornithine 8.35E+04 1.06E+05 1.32E+05 7.87E+04 8.52E+04 7.19E+04 5.57E+05 3.42E+05 3.88E+05 L-Citrulline 4.25E+04 7.92E+04 5.26E+04 4.63E+04 3.54E+04 1.98E+04 8.00E+04 5.57E+04 5.42E+04 Cadaverine 1.84E+04 1.63E+05 5.57E+04 3.47E+04 1.50E+05 5.10E+04 6.80E+04 4.08E+05 2.60E+04 Spermidine 4.14E+06 8.39E+06 4.10E+06 1.31E+07 8.38E+06 1.07E+07 2.83E+07 1.95E+07 1.02E+07 Spermine 2.47E+05 4.74E+05 2.27E+05 1.51E+06 7.41E+05 9.70E+05 6.90E+06 5.06E+06 1.64E+06 Putrescine 1.52E+05 2.03E+05 1.56E+05 1.04E+05 1.22E+05 1.43E+05 3.31E+05 2.56E+05 2.54E+05 D-Glucosamine 7.00E+04 2.13E+05 8.22E+04 1.26E+05 1.65E+05 1.76E+05 1.37E+05 1.96E+05 1.05E+05 Creatine 1.19E+07 2.22E+07 9.17E+06 6.81E+06 7.36E+06 7.10E+06 1.52E+07 1.08E+07 7.36E+06 Creatinine 1.18E+05 1.88E+05 2.55E+05 8.45E+04 1.08E+05 1.41E+05 2.68E+05 2.15E+05 2.23E+05 N-Acetylornithine 1.43E+04 4.42E+04 2.40E+04 1.27E+04 1.20E+04 2.80E+04 1.08E+05 9.26E+04 5.94E+04 Taurine 2.33E+05 2.17E+05 1.39E+05 6.31E+04 5.60E+04 6.80E+04 1.22E+05 5.85E+04 8.50E+04 L-Methionine S-oxide 4.07E+04 4.49E+04 5.42E+04 5.99E+04 4.16E+04 5.13E+04 6.02E+05 4.97E+05 2.84E+05 Indole 4.57E+05 3.97E+05 5.57E+05 5.59E+05 4.30E+05 3.99E+05 5.81E+05 5.28E+05 4.97E+05 Indole-3-acetate 1.64E+04 1.88E+04 5.45E+04 1.62E+04 1.52E+04 6.04E+03 9.60E+04 4.44E+04 2.92E+04 Sphingosine 1.98E+05 7.88E+04 2.49E+05 2.79E+05 2.16E+05 9.89E+04 2.17E+05 9.60E+04 2.16E+05 Acetylcholine 8.06E+05 1.33E+06 8.26E+05 4.36E+05 5.08E+05 5.25E+05 1.57E+06 1.43E+06 9.80E+05 Choline 2.10E+07 3.24E+07 2.13E+07 1.56E+07 1.30E+07 1.09E+07 5.07E+07 3.32E+07 2.66E+07 L-Carnitine 1.34E+06 1.73E+06 1.01E+06 1.29E+06 1.41E+06 1.28E+06 2.78E+06 2.47E+06 1.80E+06 acetyl-carnitine (acyl-C2) 5.16E+06 8.76E+06 4.84E+06 2.49E+06 2.51E+06 2.34E+06 5.04E+06 4.82E+06 2.16E+06 propionyl-carnitine (acyl-C3) 5.31E+04 1.01E+05 4.22E+04 5.35E+04 4.83E+04 5.64E+04 1.90E+05 1.32E+05 4.92E+04 butanoyl-l-carnitine (acyl-C4) 2.22E+04 1.78E+04 1.74E+04 6.15E+04 2.37E+04 2.55E+04 2.57E+04 2.68E+04 2.31E+04 acyl-C4-OH (Hydroxybutyrylcarnitine) 1.60E+05 2.68E+05 1.40E+05 4.96E+04 6.42E+04 8.26E+04 1.10E+05 1.24E+05 8.33E+04 Propionic/Propanoic acid 3.46E+05 2.69E+05 2.78E+05 3.17E+05 3.00E+05 2.57E+05 2.56E+05 2.60E+05 3.22E+05 Methylenediurea 8.09E+04 1.98E+05 6.80E+04 6.26E+04 5.54E+04 5.65E+04 1.19E+05 9.84E+04 5.29E+04 2-Deoxy-alpha-D-glucoside 6.66E+05 5.39E+05 5.93E+05 5.40E+05 4.77E+05 4.01E+05 9.63E+05 5.41E+05 6.23E+05 N-Acyl-D-mannosaminolactone 9.41E+05 1.16E+06 1.08E+06 1.65E+06 1.68E+06 1.84E+06 1.37E+06 1.63E+06 1.10E+06 9-Oxononanoic acid 5.03E+05 6.29E+05 5.13E+05 4.68E+05 4.60E+05 4.84E+05 5.22E+05 6.10E+05 5.49E+05 2-Oxo-7-methylthioheptanoic acid 2.47E+04 2.46E+04 5.81E+04 3.34E+04 1.80E+04 1.98E+04 7.68E+05 5.78E+05 5.59E+05 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. L-alanine C00041 90.0555 0.93 L-arginine C00062 175.1188 0.63 L-asparagine C00152 133.0607 0.66 L-aspartate C00049 134.0448 0.66 L-cysteine C00097 122.0272 0.67 L-glutamate C00025 148.0603 0.65 L-glutamine C00064 147.0763 0.65 L-histidine C00135 156.0766 0.62 L-leucine C00123 132.1019 0.68 L-lysine C00047 147.1126 0.60 L-methionine C00073 150.0583 0.67 L-phenylalanine C00079 166.0861 1.44 L-proline C00148 116.0708 0.67 L-serine C00065 106.0502 0.68 L-threonine C00188 120.0657 0.65 L-tryptophan C00078 205.0969 1.72 L-tyrosine C00082 182.0797 0.65 L-valine C00183 118.0864 0.66 AMP C00020 348.0693 0.68 Adenosine C00212 268.1031 0.72 Cytosine C00380 112.0508 0.78 IMP C00130 349.0533 0.78 Hypoxanthine C00262 137.0457 0.73 Urate C00366 169.0354 0.74 Nicotinamide C00153 123.0554 0.76 Thymine C00178 127.0503 0.67 Glutathione C00051 308.0902 0.83 S-Adenosyl-L-methionine C00019 399.1433 0.65 Ornithine C01602 133.0971 0.60 L-Citrulline C00327 176.1027 0.65 Cadaverine C01672 103.1233 0.60 Spermidine C00315 146.1650 0.61 Spermine C00750 203.2227 0.60 Putrescine C00134 89.1079 0.61 D-Glucosamine C00329 180.0864 0.72 Creatine C00300 132.0767 0.68 Creatinine C00791 114.0664 0.66 N-Acetylornithine C00437 175.1075 0.67 Taurine C00245 126.0220 0.70 L-Methionine S-oxide C02989 166.0531 0.85 Indole C00463 118.0653 1.89 Indole-3-acetate C00954 176.0703 1.90 Sphingosine C00319 300.2889 1.92 Acetylcholine C01996 146.1174 0.68 Choline C00114 104.1074 0.65 L-Carnitine C00318 162.1123 0.65 acetyl-carnitine (acyl-C2) C02571 204.1227 0.67 propionyl-carnitine (acyl-C3) C03017 218.1383 0.68 butanoyl-l-carnitine (acyl-C4) C02862 232.1538 1.60 acyl-C4-OH (Hydroxybutyrylcarnitine) HMDB13127 248.1486 0.67 Propionic/Propanoic acid C00163 75.0448 1.69 Methylenediurea C06381 133.0735 0.66 2-Deoxy-alpha-D-glucoside C03573 164.0679 0.58 N-Acyl-D-mannosaminolactone C03792 205.0564 0.65 9-Oxononanoic acid C16322 173.1170 1.81 2-Oxo-7-methylthioheptanoic acid C17220 191.0760 0.62 METABOLITES_END #END