#METABOLOMICS WORKBENCH AnnalauraM_20220901_062330 DATATRACK_ID:3445 STUDY_ID:ST002280 ANALYSIS_ID:AN003724 PROJECT_ID:PR001460 VERSION 1 CREATED_ON September 7, 2022, 4:59 pm #PROJECT PR:PROJECT_TITLE Oxidative phosphorylation selectively orchestrates tissue macrophage homeostasis PR:PROJECT_SUMMARY In vitro studies associated oxidative phosphorylation (OXPHOS) with PR:PROJECT_SUMMARY anti-inflammatory macrophages, while pro-inflammatory macrophages rely on PR:PROJECT_SUMMARY glycolysis. However, the metabolic needs of macrophages in tissues (TMFs) to PR:PROJECT_SUMMARY fulfil their homeostatic activities are incompletely understood. Here, we PR:PROJECT_SUMMARY identified OXPHOS as highly discriminating process among TMFs from different PR:PROJECT_SUMMARY tissues in homeostasis by analysis of RNAseq data, in both human and mouse. PR:PROJECT_SUMMARY Impairing OXPHOS in TMFs via Tfam deletion differentially affected TMF PR:PROJECT_SUMMARY populations. Tfam deletion resulted in reduction of alveolar macrophages (AMs) PR:PROJECT_SUMMARY due to impaired lipid-handling capacity, leading to increased cholesterol PR:PROJECT_SUMMARY content and cellular stress, causing cell cycle arrest in vivo. In obesity, Tfam PR:PROJECT_SUMMARY depletion selectively ablated pro-inflammatory lipid-handling white adipose PR:PROJECT_SUMMARY tissue macrophages (WAT-MFs), preventing insulin resistance and hepatosteatosis. PR:PROJECT_SUMMARY Thus, OXPHOS, rather than glycolysis, distinguishes TMF populations and is PR:PROJECT_SUMMARY critical for the maintenance of TMFs with a high lipid-handling activity, PR:PROJECT_SUMMARY including pro-inflammatory WAT-MFs. This could provide a selective therapeutic PR:PROJECT_SUMMARY targeting tool. PR:INSTITUTE Spanish National Center for Cardiovascular Research (CNIC) PR:DEPARTMENT Novel mechanisms of atherosclerosis PR:LABORATORY Immunobiology PR:LAST_NAME Mastrangelo PR:FIRST_NAME Annalaura PR:ADDRESS Calle de Melchor Fernández Almagro, 3, Centro Nacional de Investigaciones PR:ADDRESS Cardiovasculares PR:EMAIL annalaura.mastrangelo@cnic.es PR:PHONE (+34) 914531200 #STUDY ST:STUDY_TITLE Oxidative phosphorylation selectively orchestrates tissue macrophage homeostasis ST:STUDY_TYPE Observational study ST:STUDY_SUMMARY In vitro studies associated oxidative phosphorylation (OXPHOS) with ST:STUDY_SUMMARY anti-inflammatory macrophages, while pro-inflammatory macrophages rely on ST:STUDY_SUMMARY glycolysis. However, the metabolic needs of macrophages in tissues (TMFs) to ST:STUDY_SUMMARY fulfil their homeostatic activities are incompletely understood. Here, we ST:STUDY_SUMMARY identified OXPHOS as highly discriminating process among TMFs from different ST:STUDY_SUMMARY tissues in homeostasis by analysis of RNAseq data, in both human and mouse. ST:STUDY_SUMMARY Impairing OXPHOS in TMFs via Tfam deletion differentially affected TMF ST:STUDY_SUMMARY populations. Tfam deletion resulted in reduction of alveolar macrophages (AMs) ST:STUDY_SUMMARY due to impaired lipid-handling capacity, leading to increased cholesterol ST:STUDY_SUMMARY content and cellular stress, causing cell cycle arrest in vivo. In obesity, Tfam ST:STUDY_SUMMARY depletion selectively ablated pro-inflammatory lipid-handling white adipose ST:STUDY_SUMMARY tissue macrophages (WAT-MFs), preventing insulin resistance and hepatosteatosis. ST:STUDY_SUMMARY Thus, OXPHOS, rather than glycolysis, distinguishes TMF populations and is ST:STUDY_SUMMARY critical for the maintenance of TMFs with a high lipid-handling activity, ST:STUDY_SUMMARY including pro-inflammatory WAT-MFs. This could provide a selective therapeutic ST:STUDY_SUMMARY targeting tool. ST:INSTITUTE Spanish National Center for Cardiovascular Research (CNIC) ST:DEPARTMENT Novel mechanisms of atherosclerosis ST:LABORATORY Immunobiology ST:LAST_NAME Mastrangelo ST:FIRST_NAME Annalaura ST:ADDRESS Calle de Melchor Fernández Almagro, 3, Centro Nacional de Investigaciones ST:ADDRESS Cardiovasculares ST:EMAIL annalaura.mastrangelo@cnic.es ST:PHONE (+34) 914531200 ST:NUM_GROUPS 2 #SUBJECT SU:SUBJECT_TYPE Mammal 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 KO1 KO1 Genoptype:Mutant RAW_FILE_NAME=KO1.mzML SUBJECT_SAMPLE_FACTORS KO1 KO1R Genoptype:Mutant RAW_FILE_NAME=KO1R.mzML SUBJECT_SAMPLE_FACTORS KO3 KO3 Genoptype:Mutant RAW_FILE_NAME=KO3.mzML SUBJECT_SAMPLE_FACTORS KO3 KO3R Genoptype:Mutant RAW_FILE_NAME=KO3R.mzML SUBJECT_SAMPLE_FACTORS KO2 KO2R Genoptype:Mutant RAW_FILE_NAME=KO2R.mzML SUBJECT_SAMPLE_FACTORS WT1 WT1 Genoptype:Wild_type RAW_FILE_NAME=WT1.mzML SUBJECT_SAMPLE_FACTORS WT1 WT1R Genoptype:Wild_type RAW_FILE_NAME=WT1R.mzML SUBJECT_SAMPLE_FACTORS WT2 WT2 Genoptype:Wild_type RAW_FILE_NAME=WT2.mzML SUBJECT_SAMPLE_FACTORS WT2 WT2R Genoptype:Wild_type RAW_FILE_NAME=WT2R.mzML SUBJECT_SAMPLE_FACTORS WT3 WT3 Genoptype:Wild_type RAW_FILE_NAME=WT3.mzML SUBJECT_SAMPLE_FACTORS WT3 WT3R Genoptype:Wild_type RAW_FILE_NAME=WT3R.mzML #COLLECTION CO:COLLECTION_SUMMARY Mouse colonies were bred at the CNIC under specific pathogen-free conditions and CO:COLLECTION_SUMMARY on C57BL/6 background. Tfamf/f (Larsson et al., 1998) mice were kindly provided CO:COLLECTION_SUMMARY by Nils-Göran Larsson (Max Planck Institute for Biology of Ageing, Cologne, CO:COLLECTION_SUMMARY Germany). All floxed mouse lines were crossed with CD11cCre mice (Caton et al., CO:COLLECTION_SUMMARY 2007). Mice were group-housed, have not been used in previous procedures and CO:COLLECTION_SUMMARY were fed standard chow. Littermates of the same sex were randomly assigned to CO:COLLECTION_SUMMARY experimental groups. Male and female mice were used for all experiments. Mice CO:COLLECTION_SUMMARY with 6–10-weeks (adult) were used for the experiment. Bronchoalveolar lavage CO:COLLECTION_SUMMARY (BAL) was performed by inserting a venal catheter (BD) into the trachea and 3-10 CO:COLLECTION_SUMMARY washes with 0.3-1 ml FACS buffer to harvest BAL cells. CO:SAMPLE_TYPE Bronchoalveolar lavage CO:COLLECTION_METHOD Bronchoalveolar lavage (BAL) was performed by inserting a venal catheter (BD) CO:COLLECTION_METHOD into the trachea and 3-10 washes with 0.3-1 ml FACS buffer to harvest BAL cells. CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Mice were group-housed and were fed standard chow. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY One million CD45+ F4/80+ CD11c+ FACS-sorted alveolar macrophages (AMs) from the SP:SAMPLEPREP_SUMMARY bronchoalveolar lavage (BAL) of adult Tfamf/f and CD11c∆Tfam mice were SP:SAMPLEPREP_SUMMARY collected. Each sample was generated by merging FACS-sorted AMs from 13 to 30 SP:SAMPLEPREP_SUMMARY mice. Quality Control (QC) samples (n=4) were prepared by pooling equal volumes SP:SAMPLEPREP_SUMMARY of cell extracts from each sample by following the same protocols used for the SP:SAMPLEPREP_SUMMARY subject samples. Samples were subjected to two freeze–thaw cycles for SP:SAMPLEPREP_SUMMARY metabolism quenching and complete metabolite extraction, specifically by placing SP:SAMPLEPREP_SUMMARY the samples at -80ºC for 15 min and thawing them on ice for 10 min with brief SP:SAMPLEPREP_SUMMARY vortex-mixing. The samples were then centrifuged at 20,000 xg at 4°C for 10 min SP:SAMPLEPREP_SUMMARY and the supernatant collected. The supernatant was evaporated to dryness SP:SAMPLEPREP_SUMMARY (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA) and SP:SAMPLEPREP_SUMMARY derivatized with 10 μl O-methoxyamine hydrochloride (15mg/mL) in pyridine and SP:SAMPLEPREP_SUMMARY 10 μl N,O-bis(trimethylsilyl)trifluoroacetamide in 1% trimethylchlorosilane. SP:SAMPLEPREP_SUMMARY Finally, 100 μl of heptane containing 10 ppm of 4-nitrobenzoic acid (IS) was SP:SAMPLEPREP_SUMMARY used as internal standard to monitor sample injection #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Derivative samples (2 μL) were injected into a GC column DB5–MS (30 m length, CH:CHROMATOGRAPHY_SUMMARY 0.250 mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with a CH:CHROMATOGRAPHY_SUMMARY pre–column (10 m J&W integrated with Agilent 122–5532G). The temperature CH:CHROMATOGRAPHY_SUMMARY gradient was programmed at 60 °C (held for 1 min), with a ramping increase rate CH:CHROMATOGRAPHY_SUMMARY of 10 °C/min up to 325°C (held for 10 min). The total analysis time was 37.5 CH:CHROMATOGRAPHY_SUMMARY min. Two analytical replicates for each sample were injected. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7250 GC/Q-TOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The EI source was operated at 70 eV whereas the mass spectrometer operated in MS:MS_COMMENTS the scan mode over a mass range of m/z 50–600. Metabolite deconvolution and MS:MS_COMMENTS identification were carried out using Agilent MassHunter Unknowns Analysis MS:MS_COMMENTS version B.07.00, then, data was aligned in Agilent Mass Profiler Professional MS:MS_COMMENTS version B.12.1 and exported to Agilent MassHunter Quantitative Analysis version MS:MS_COMMENTS B.07.00. Metabolites were identified by comparing their retention time, MS:MS_COMMENTS retention index and mass fragmentation patterns with those available in an MS:MS_COMMENTS in-house library including both the NIST mass spectral database (version 2017) MS:MS_COMMENTS and Fiehn RTL library (version 2008). The different derivatives that were MS:MS_COMMENTS generated from the silylated compounds were unified by summing the abundance of MS:MS_COMMENTS all derivatives from the same metabolite. Finally, the median relative area of MS:MS_COMMENTS the two analytical replicates of the same sample was computed and used for MS:MS_COMMENTS subsequent statistical analysis. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative abundance MS_METABOLITE_DATA_START Samples KO1 KO1R KO2R KO3 KO3R WT1 WT1R WT2 WT2R WT3 WT3R Factors Genoptype:Mutant Genoptype:Mutant Genoptype:Mutant Genoptype:Mutant Genoptype:Mutant Genoptype:Wild_type Genoptype:Wild_type Genoptype:Wild_type Genoptype:Wild_type Genoptype:Wild_type Genoptype:Wild_type Monoglycerol(16:0) 1544.39 1859.33 1456 1157.3 1261.46 1419.52 1293.99 901.51 918.9 1196.17 1196.71 6-Deoxyglucose 262.13 389.71 311 282.84 308.35 168 123.79 230.96 281.02 149.91 147.28 Alanine 1813.68 2039.41 2171 2773.04 2059.82 1519.24 1514.69 1253.32 1482.19 1732.83 1532.32 Arachidic acid 168.3 187.14 190 194.68 208.16 199.6 176.88 194.92 183.48 210.21 134.39 Arachidonic acid 1279.98 1250.58 1190 1159.05 1072.16 939.55 893.21 928.93 854.97 1135.87 1011.01 Aspartic acid 1685.63 1970.46 2105 2442.26 2322.53 1227.66 1318.78 877.62 949.75 998.36 930.16 Cholesterol 204100.3355 204950.121 186654 167792.5424 169773.9365 110034.1367 108534.1513 112531.2553 109774.6827 107986.8689 105313.9675 Citric acid 1152.92 1191.77 1151 1091 1169.47 869.22 967.57 1064.91 1226.35 719.11 703.96 Decanoic acid 4376.64 4255.23 4586 5029.79 4682.35 5017.78 5084.47 4651.73 4548 4969.81 4669.5 Eicosapentaenoic acid 1452.72 1482.11 5667 9989.79 9745.03 1709.84 1655.43 1431 1495.2 2945.34 3065.06 Fumaric acid 760.59 711.51 738 717.96 761.8 488.34 518.81 470.4 407.49 496.5 550.58 Glucose 367.55 460.57 458 500.4 503.55 301.07 343.37 270.68 335.82 306.02 218.57 Glutamic acid 1433.24 1619.86 1630 1786.23 1682.52 879.97 869.7 707.16 959.58 886.91 710.34 Glycerol 3017.88 2722.1 2664 2373.91 2542.64 1797.08 1690.93 2108.47 2361.38 2798.67 2627.65 Glycerol 1-phosphate 865.59 782.07 758 669.29 714.44 539.55 486.83 411.74 374.98 622.46 424.1 Glycine 1827.29 1825.22 2814 2289.4 2046.15 1335.91 1154.21 1733.62 1819.12 1549.25 1229.02 Isocitric acid 385.45 478.36 450 428.27 507 303.91 280.2 175.98 166.46 223.6 183.62 Isoleucine 1016.56 1052.71 1047 1065.94 1051.58 594.07 648.19 516.06 504.9 534.12 622.55 Lactic acid 1582.21 1839.04 1754 1829.51 1763.42 1525.4 1490.7 1574.46 1609.21 1666.47 1886.25 Leucine 69981.01 71142.5 58864 47440.08 46891.91 66295.54 67064.46 33895.56 34665.72 49295.33 49228.22 Malic acid 229.9 227.42 221 227.61 200.96 149.51 141.7 67.85 107.65 143.43 132.3 Myoinositol 1019.712435 904.0846988 948 932.1662191 937.8526844 619.5179771 728.9580921 428.1399165 532.0223491 369.8407219 333.3971281 Myoinositol 6phosphate 756.15 740.03 707 674.53 656.42 513.95 497.54 506.3 406.52 499.46 401.91 Myristic acid 1116.254952 1160.903555 1106 1094.351775 1053.158901 1196.207033 1031.864329 1148.726676 1050.623068 1185.885469 1129.445179 Oleic Acid 1689.06 1620.88 1471 1312.49 1263.04 1427.58 1382.91 1199.33 1253.75 1265.91 1185.33 Palmitelaidic acid 643.5690365 654.7631298 616 533.0411176 630.6573462 479.494271 440.2855699 530.3337626 577.268463 608.5112678 475.5485659 Palmitic acid 69113.56 72139.78 65577 59322.17 61733.45 70269.62 70379.96 67594.52 67941.95 67367.65 65340.71 Palmitoleic acid 434.01 476.15 366 245.25 309.93 355.59 349.77 261.21 259.02 344.48 279.03 Proline 2159.32 2418.54 2498 2852.16 2560.78 1640.46 1706.29 1514.53 1687.94 1747.2 1756.99 Pyruvic acid 7430.34 7848.19 7794 7891.14 8007.23 8449.62 8458.5 7996.7 8932.16 7861.19 7687 Serine 1095.2 1262.48 1338 1514.17 1482.14 1100.43 1176.12 962.86 1005.65 1014.98 893.4 Stearic acid 8595.44 8510.01 9314 9825.12 10325.74 10825.13 10845.77 9895.13 9988.18 9647.56 9925.36 Succinic acid 1269.52 1397.58 1322 1098.14 1522.73 1233.5 1446.88 1171.77 1051.59 1097.97 1529.97 Threonine 433.44 527.75 503 557.67 492.75 321.77 274.6 258.1 212.95 325.18 276.95 Valine 2055.47 2146.87 2102 2107.39 2096.82 1242.2 1024.03 841.44 1013.68 1200.12 1209.19 Cysteine 822.33 811.32 889 1130.78 954.7 596.6 388.15 336.55 444.71 557.93 525.47 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pubchem ID KEGG ID Retention_time Target_Ion Retention_Index Monoglycerol(16:0) 14900 - 23.43 371 2581 6-Deoxyglucose 93579 C00586 15.54 117.07 1998 Alanine 5950 C00041 7.404 116 777.72 Arachidic acid 10467 C06425 22.33 369.30 2385 Arachidonic acid 444899 C00219 21.681 91 2417 Aspartic acid 5960 C00049 12.101 160 1102.84 Cholesterol 5997 C00187 27.488 329 2826.78 Citric acid 311 C00158 16.458 273 1493.93 Decanoic acid 2969 C01571 16.63 229.20 1391 Eicosapentaenoic acid 446284 C06428 23.86 361.20 2425 Fumaric acid 444972 C00122 10.868 245 1025.3 Glucose 5793 C00031 17.36 319.20 1579.97 Glutamic acid 33032 C00025 14.256 246 1297.52 Glycerol 753 C00116 9.806 205 949.79 Glycerol 1-phosphate 3707990 C00623 15.879 357 1439.25 Glycine 750 C00037 7.66 102 1002 Isocitric acid 1198 C00311 16.897 273 1497.48 Isoleucine 6306 C00407 10.084 158 969.56 Lactic acid 612 C00186 6.794 147 732.96 Leucine 6106 C00123 7.96 86 831.85 Malic acid 525 C00711 12.433 233 1164.45 Myoinositol 892 C00137 19.29 305.10 2194 Myoinositol 6phosphate 161368 C01284 22.12 318.20 2386 Myristic acid 11005 C06424 16.821 117 1524.35 Oleic Acid 445639 C00712 20.381 339 1892.45 Palmitelaidic acid 5282745 - 18.65 311.20 1995 Palmitic acid 985 C00249 18.793 117 1720.26 Palmitoleic acid 445638 C08362 18.554 311 1701.83 Proline 145742 C00148 10.162 142 975.16 Pyruvic acid 1060 C00022 6.643 174 721.67 Serine 5951 C00065 10.991 204 1040.14 Stearic acid 5281 C01530 20.608 341 1916.02 Succinic acid 1110 C00042 10.387 247 989.72 Threonine 6288 C00188 11.329 218 1063.93 Valine 6287 C00183 7.219 72 762.88 Cysteine 5862 C00097 13.43 218 1230.5 METABOLITES_END #END