#METABOLOMICS WORKBENCH juliehaines_20241126_073648 DATATRACK_ID:5405 STUDY_ID:ST003610 ANALYSIS_ID:AN005933 PROJECT_ID:PR002231 VERSION 1 CREATED_ON December 2, 2024, 3:15 pm #PROJECT PR:PROJECT_TITLE Macrophages recycle phagocytosed bacteria to fuel immunometabolic responses PR:PROJECT_SUMMARY Macrophages specialize in phagocytosis, a cellular process that eliminates PR:PROJECT_SUMMARY extracellular matter, including microbes, through internalization and PR:PROJECT_SUMMARY degradation. Despite the critical role of phagocytosis during bacterial PR:PROJECT_SUMMARY infection, the fate of phagocytosed microbial cargo and its impact on host cell PR:PROJECT_SUMMARY is poorly understood. Here, we reveal that ingested bacteria constitute an PR:PROJECT_SUMMARY alternative nutrient source that skews immunometabolic host responses. Tracing PR:PROJECT_SUMMARY stable isotope-labelled bacteria, we found that phagolysosomal degradation of PR:PROJECT_SUMMARY bacteria provides carbon atoms and amino acids that are recycled into various PR:PROJECT_SUMMARY metabolic pathways, including glutathione and itaconate biosynthesis, and PR:PROJECT_SUMMARY satisfy macrophage bioenergetic needs. Metabolic recycling of PR:PROJECT_SUMMARY microbially-derived nutrients is regulated by the nutrient sensing mTORC1 and PR:PROJECT_SUMMARY intricately tied to microbial viability. Dead bacteria, as opposed to live ones, PR:PROJECT_SUMMARY are enriched in cyclic- adenosine monophosphate (AMP), sustain the cellular AMP PR:PROJECT_SUMMARY pool and subsequently activate AMP protein kinase (AMPK) to inhibit mTORC1. PR:PROJECT_SUMMARY Consequently, killed bacteria strongly fuel metabolic recycling and support PR:PROJECT_SUMMARY macrophage survival, but elicit decreased reactive oxygen species (ROS) PR:PROJECT_SUMMARY production and a reduced IL-1β secretion compared to viable bacteria. These PR:PROJECT_SUMMARY results reveal a novel insight into the fate of engulfed microbes and highlights PR:PROJECT_SUMMARY a microbial viability-associated metabolite that triggers host metabolic and PR:PROJECT_SUMMARY immune responses. Our findings hold promise for shaping immunometabolic PR:PROJECT_SUMMARY intervention in various immune-related pathologies. PR:INSTITUTE University of Colorado Anschutz Medical Campus PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Johan Garaude (INSERM, PR:LABORATORY Fr) 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 13C-tracing metabolomics of peritoneal macrophages 1hour and 2hours after ST:STUDY_TITLE engulfment of 13C-labeled heat-killed E coli ST:STUDY_SUMMARY 13C-tracing analysis at 1h and 2h of peritoneal macrophages isolated from ST:STUDY_SUMMARY C57BL6/N mice injected with uniformly 13C-labelled and DAPI labeled heat-killed ST:STUDY_SUMMARY E. coli. ST:INSTITUTE University of Colorado Anschutz Medical Campus 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:GENDER Not applicable #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 - AA83PM-1_r19- Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-1_r19- SUBJECT_SAMPLE_FACTORS - AA83PM-2_r10- Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-2_r10- SUBJECT_SAMPLE_FACTORS - AA83PM-3_r17- Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-3_r17- SUBJECT_SAMPLE_FACTORS - AA83PM-4_r15- Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-4_r15- SUBJECT_SAMPLE_FACTORS - AA83PM-5_r9- Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-5_r9- SUBJECT_SAMPLE_FACTORS - AA83PM-6_r3- Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-6_r3- SUBJECT_SAMPLE_FACTORS - AA83PM-7_r16- Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-7_r16- SUBJECT_SAMPLE_FACTORS - AA83PM-8_r5- Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-8_r5- SUBJECT_SAMPLE_FACTORS - AA83PM-9_r13- Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-9_r13- SUBJECT_SAMPLE_FACTORS - AA83PM-10_r6- Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-10_r6- SUBJECT_SAMPLE_FACTORS - AA83PM-11_r2- Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-11_r2- SUBJECT_SAMPLE_FACTORS - AA83PM-12_r18- Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-12_r18- SUBJECT_SAMPLE_FACTORS - AA83PM-13_r7- Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-13_r7- SUBJECT_SAMPLE_FACTORS - AA83PM-14_r8- Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-14_r8- SUBJECT_SAMPLE_FACTORS - AA83PM-15_r12- Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-15_r12- SUBJECT_SAMPLE_FACTORS - AA83PM-16_r14- Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-16_r14- SUBJECT_SAMPLE_FACTORS - AA83PM-17_r4- Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-17_r4- SUBJECT_SAMPLE_FACTORS - AA83PM-18_r11- Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-18_r11- SUBJECT_SAMPLE_FACTORS - AA83PM-19_r1- Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells RAW_FILE_NAME(raw file name)=AA83PM-19_r1- #COLLECTION CO:COLLECTION_SUMMARY Cells from the peritoneal cavity were harvested with cold PBS. Phagocytic cells CO:COLLECTION_SUMMARY (DAPI+ cells) were sorted by flow cytometry, frozen as dry cell pellet, and CO:COLLECTION_SUMMARY stored at -80˚C until processing. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY Preparation of macrophages: Mice were injected or not with 0.5 ml of TR:TREATMENT_SUMMARY thioglycolate medium to increase peritoneal macrophage abundance. Four days TR:TREATMENT_SUMMARY later, mice were injected with 1E9 killed U-[13C]Bacteria previously labeled TR:TREATMENT_SUMMARY with DAPI. One or two hours later, peritoneal cells were harvested, and sorted TR:TREATMENT_SUMMARY by flow cytometry based on DAPI. Preparation of viable and killed TR:TREATMENT_SUMMARY U-[13C]Bacteria: ThyA- E. coli were grown overnight with shaking in LB TR:TREATMENT_SUMMARY supplemented with thymidine (500 ug/ml) and trimethoprim (50 ug/ml), diluted TR:TREATMENT_SUMMARY 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. TR:TREATMENT_SUMMARY Bacteria were washed with phosphate buffer saline (PBS) to remove LB salts TR:TREATMENT_SUMMARY before addition to cells. For labeling of bacteria, 10 ul of an overnight TR:TREATMENT_SUMMARY cultured of thyA- E. coli was added to 20 ml of a filtered M9 minimal medium TR:TREATMENT_SUMMARY salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M TR:TREATMENT_SUMMARY CaCl2, 500 ug/ml thymidine, 50 ug/ml trimethoprim, and 0.5% U-[13C] glucose TR:TREATMENT_SUMMARY (Campro Scientific). Bacteria were grown for 72h, washed with PBS and subjected TR:TREATMENT_SUMMARY to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for TR:TREATMENT_SUMMARY 60-90 min. Bacteria were kept at 4˚C until use. Efficient killing was confirmed TR:TREATMENT_SUMMARY by overnight plating on LB-agar plates. For DAPI labelling, killed bacteria were TR:TREATMENT_SUMMARY incubated with 0.2 ug/ml of DAPI in PBS for 5min and washed 3 times with cold TR:TREATMENT_SUMMARY PBS. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from frozen pellets were extracted at 4e6 cells per mL using ice SP:SAMPLEPREP_SUMMARY cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 SP:SAMPLEPREP_SUMMARY degrees C followed by centrifugation as described for 10 min at 18,000 g. SP:SAMPLEPREP_SUMMARY Supernatants were maintained at 4°C until analysis that same day. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Negative C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 95% water/5% acetonitrile; 1 mM ammonium acetate CH:SOLVENT_B 95% acetonitrile/5% water; 1 mM ammonium acetate CH:FLOW_GRADIENT 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min CH:FLOW_GRADIENT 100-0% B, 3-5 min hold at 0% B CH:FLOW_RATE 450 uL/min CH:COLUMN_TEMPERATURE 45 CH:SAMPLE_INJECTION 10 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 120 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 MS:MS_COMMENTS m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) MS:MS_COMMENTS detection duration 20 msec, source voltage 2.0 kV, capillary temperature 320 C, MS:MS_COMMENTS vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all MS:MS_COMMENTS nitrogen). Data converted to mzXML using RawConverter. Metabolites were MS:MS_COMMENTS annotated and integrated using Maven in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples AA83PM-1_r19- AA83PM-2_r10- AA83PM-3_r17- AA83PM-4_r15- AA83PM-5_r9- AA83PM-6_r3- AA83PM-7_r16- AA83PM-8_r5- AA83PM-9_r13- AA83PM-10_r6- AA83PM-11_r2- AA83PM-12_r18- AA83PM-13_r7- AA83PM-14_r8- AA83PM-15_r12- AA83PM-16_r14- AA83PM-17_r4- AA83PM-18_r11- AA83PM-19_r1- Factors Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:1h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:13C-labelled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells Killed E coli labeling:Unlabeled KEC | Timepoint:2h | Sample source:cells ADP 775710.69 1229823.62 1488447.62 686576.75 955402.81 413119.16 259618.81 369281.75 711871.31 331348.91 281276.09 446111.03 269246.41 253227.25 334667.25 230293.33 259736.05 229945.5 287624.31 AMP 1139820.62 1600551.5 2011828.88 1397026.88 1524229.5 437166.91 424943.41 792692 1191836.88 626192.44 344703.78 654159.44 356164.75 314061.5 1934660.62 3624747.25 1134967.88 4834486 1036974.56 Adenine 338989.25 245081.27 267469.72 327352.91 288457.28 432892.62 248050.61 309789.97 272683.62 656019.31 542233.06 887234.31 758108.94 759449.81 873062.81 802822.19 651217.31 745916 523926.91 Adenine 13C1 0 0 0 3160.25995 0 0 0 0 0 0 5058.5717 0 0 0 0 0 0 0 0 Citrate 27128682 45101092 43615888 21598714 26824802 5108305.5 4351005.5 4688781.5 6231690.5 4361508.5 1483650 1864554.88 1704246.12 1598007.5 2073350 1935391.88 2785719.75 2620453.25 2766194 Citrate 13C1 1864792.988 1850210.428 0 0 750131.818 0 2302.797 41550.891 100228.147 17365.529 66575.4 188182.7879 53047.06608 54365.985 14565.48 43254.58592 56357.6865 69484.7255 0 Citrate 13C2 1674249.081 1929285.393 307759.0319 175353.5318 870531.1625 41152.49124 6124.970042 18838.49779 49556.51618 96115.97897 122341.8806 164600.2789 147405.6039 114799.0493 22235.7474 37127.42297 107156.6148 52224.01564 62320.44894 Citrate 13C3 232077.14 226969.92 30800.25 19096.57 94495.16 0 0 0 14870.15 10520.12 50148.52 45329.86 16889.86 15282.44 0 0 3796.89 7610 2129.33 Citrate 13C4 644353.44 731912.19 2067.02 61260.79 436853.97 0 0 674.82 21363.8 89500.21 116590.54 157959.72 116474.12 76287.95 0 2291.59 19156.27 8857.6 8093.99 2-Oxoglutarate 57672.21 226745.27 249799.92 77013.72 53871.75 7437.14 0 19837.63 24908.78 71717.28 36736.71 39378.42 41125.47 21726.6 99641.04 51896.59 39307.18 26879.76 34181.55 Succinate 493472.25 773872.69 789158.19 341393.59 700532.5 275527.66 171200.8 264057.69 389344.97 479684.91 382421.22 367002.75 335504.25 358063.41 283666.34 255686.52 245006.12 296214.97 234878.92 Fumarate 5466648 12300443 12967037 6378663.5 12645368 115167.13 115248.4 214981.08 297719.84 6195821.5 3246315.25 2441518 2447697.25 2086989.38 4390945.5 1683302.62 2836062.75 1925170 2841338 Fumarate 13C1 15671.658 0 0 18778.586 0 0 2902.1704 0 0 0 0 7460.978 19637.841 18486.51728 0 0 0 0 0 Fumarate 13C2 13194.95947 0 0 1659.137464 0 0 0 0 0 0 9107.383676 10370.38115 5081.418124 15400.17856 0 0 0 0 0 Malate 5757498.5 8902595 11243643 4237363.5 6673000 1550770.12 1853419.5 1775661.62 2620554.75 3326103.25 2513006.25 2461847.25 2073137.12 2293846 2180637.75 1807180.38 2408826.75 2138661.25 1946797.62 Malate 13C1 391501.876 428800.57 18610.958 0 105230.91 0 0 0 0 111934.597 183619.415 158921.381 181462.4067 125198.726 7927.729 50036.71328 32545.203 32946.065 41730.88472 Malate 13C2 350151.2329 300358.9161 102204.5172 13714.02426 200066.492 14424.07905 12186.26985 16741.2591 19645.876 132510.9341 323154.4099 216571.0637 335199.0265 253271.0041 31499.61532 28619.12878 35095.15141 44337.44182 53557.52981 Malate 13C4 118819.15 169336.25 1134.68 11139.31 92472.07 830.77 946.06 0 0 282850.03 475762.5 349379.78 457072.25 448343.72 7788.37 15274.14 34533.22 15201.04 17352.58 Itaconate 1667004.38 2018256.5 1946360.88 1295182.88 6721433.5 92721.88 109851.66 130647.31 168488.39 344910.66 269142.72 263462.5 191939.31 199644.05 494527.16 230638.58 178002.31 167447.23 224432.86 Itaconate 13C1 69944.8491 80695.3425 0 0 197474.7175 0 0 0 0 0 0 9321.6425 11692.48795 0 0 0 0 0 0 Itaconate 13C2 97759.71175 51017.45409 6840.388338 14111.61179 155034.4337 2781.956313 1602.538729 1271.061887 903.6726203 488.6552535 3974.713272 11758.53594 4595.003587 2470.626749 3127.035341 2226.788296 3627.353012 4704.252129 4046.790599 Glutathione 10736893 9334501 10892585 10633553 6819784.5 3426732.75 2624270 1056896.5 6303504.5 13526681 10558747 10463117 8282046 8688515 19472130 17233598 16435035 18519262 17120112 Glutathione 13C1 65506.77 0 59662.77 0 50188.645 0 11733.96 8089.925 0 0 0 50100.88 0 0 82188.95 0 0 0 0 Glutathione 13C2 358737.6847 181824.5379 0 0 142648.1676 690.523725 0 0 0 0 160196.9113 186585.1943 145984.3634 123522.6085 0 0 0 0 0 Glutathione 13C3 309994.84 110632.88 0 0 34382.04 0 0 0 0 37926.27 133408.64 137495.73 72133.92 52020.8 15106.34 25893.14 23158.97 20201.69 15597.71 Glutathione 13C4 29094.42 10896.04 0 0 0 0 0 0 0 12434.61 33490.83 30765.54 42114.44 9637.58 14890.55 46340.45 3852.81 20983.97 4624.17 Glutathione 13C5 136820.45 0 0 0 0 0 0 0 0 44864.59 134755.11 133256.56 134830.92 82265.35 0 0 11778.89 0 3490.03 Glutathione 13C7 0 0 0 0 0 7002.4 0 0 0 6086.47 47995.45 33864.5 47990.97 45416.33 4686.19 5794.91 4127.29 5135.13 2695.04 Glutathione 13C10 0 19660.25 14221.15 12904.36 27478.48 0 0 0 0 9200.89 43186.11 6198.84 7618.67 11639.09 5118.15 9690.62 3346.78 0 0 Glutathione disulfide 29565.29 107213.85 112784.65 32308.99 0 46443.62 52467.28 177599.81 165055.03 33563.25 37516.15 16128.4 21234.74 46040.85 22013.52 13232.04 8367.81 15806.85 17898.92 Glutathione disulfide 13C1 29565.29 102888.595 109655.997 29470.0308 0 46443.62 52467.28 177599.81 165055.03 31539.0542 28015.2058 14764.6552 19558.6326 43480.2502 20887.527 11100.1036 7631.5184 15806.85 17898.92 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID mz rt ADP C00008 426.022095 0.564 AMP C00020 346.055939 0.622 Adenine C00147 134.047318 0.679 Adenine 13C1 C00147 135.05069 0.681 Citrate C00158 191.019608 0.543 Citrate 13C1 C00158 192.023117 0.542 Citrate 13C2 C00158 193.025818 0.545 Citrate 13C3 C00158 194.029663 0.565 Citrate 13C4 C00158 195.032059 0.55 2-Oxoglutarate C00026 145.014191 0.619 Succinate C00042 117.019409 0.556 Fumarate C00122 115.003784 0.566 Fumarate 13C1 C00122 116.006981 0.568 Fumarate 13C2 C00122 117.009911 0.562 Malate C00149 133.014191 0.547 Malate 13C1 C00149 134.017593 0.554 Malate 13C2 C00149 135.018997 0.558 Malate 13C4 C00149 137.028915 0.53 Itaconate C00490 129.019424 0.602 Itaconate 13C1 C00490 130.02269 0.589 Itaconate 13C2 C00490 131.023956 0.615 Glutathione C00051 306.076477 0.591 Glutathione 13C1 C00051 307.07782 0.591 Glutathione 13C2 C00051 308.082001 0.593 Glutathione 13C3 C00051 309.085541 0.594 Glutathione 13C4 C00051 310.087769 0.608 Glutathione 13C5 C00051 311.093353 0.593 Glutathione 13C7 C00051 313.098846 0.634 Glutathione 13C10 C00051 316.114136 0.627 Glutathione disulfide C00127 611.144592 0.531 Glutathione disulfide 13C1 C00127 612.148071 0.537 METABOLITES_END #END