#METABOLOMICS WORKBENCH juliehaines_20241126_074650 DATATRACK_ID:5406 STUDY_ID:ST003611 ANALYSIS_ID:AN005935 PROJECT_ID:PR002231 VERSION 1 CREATED_ON December 5, 2024, 7:54 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 after engulfment of ST:STUDY_TITLE 13C-labeled heat-killed E coli vs untreated peritoneal macrophages ST:STUDY_SUMMARY 13C-tracing analysis of peritoneal resident macrophages isolated from C57BL6/N ST:STUDY_SUMMARY mice injected or not with uniformly 13C-labelled heat-killed E. coli, 1 hours ST:STUDY_SUMMARY post-infection. 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 - AA89-53_r97- treatment:control | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-53_r97- SUBJECT_SAMPLE_FACTORS - AA89-54_r38- treatment:control | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-54_r38- SUBJECT_SAMPLE_FACTORS - AA89-55_r110- treatment:control | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-55_r110- SUBJECT_SAMPLE_FACTORS - AA89-56_r96- treatment:control | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-56_r96- SUBJECT_SAMPLE_FACTORS - AA89-57_r41- treatment:control | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-57_r41- SUBJECT_SAMPLE_FACTORS - AA89-58_r71- treatment:Ecoli treated | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-58_r71- SUBJECT_SAMPLE_FACTORS - AA89-59_r64- treatment:Ecoli treated | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-59_r64- SUBJECT_SAMPLE_FACTORS - AA89-60_r95- treatment:Ecoli treated | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-60_r95- SUBJECT_SAMPLE_FACTORS - AA89-61_r19- treatment:Ecoli treated | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-61_r19- SUBJECT_SAMPLE_FACTORS - AA89-62_r17- treatment:Ecoli treated | Sample source:murine peritoneal macrophages RAW_FILE_NAME(raw file name)=AA89-62_r17- #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 killed U-[13C]Bacteria: ThyA- E. TR:TREATMENT_SUMMARY coli were grown overnight with shaking in LB supplemented with thymidine (500 TR:TREATMENT_SUMMARY ug/ml) and trimethoprim (50 ug/ml), diluted 1/40, and grown until log-phase TR:TREATMENT_SUMMARY [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with TR:TREATMENT_SUMMARY phosphate buffer saline (PBS) to remove LB salts before addition to cells. For TR:TREATMENT_SUMMARY labeling of bacteria, 10 ul of an overnight cultured of thyA- E. coli was added TR:TREATMENT_SUMMARY to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented TR:TREATMENT_SUMMARY with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, 500 ug/ml thymidine, 50 ug/ml TR:TREATMENT_SUMMARY trimethoprim, and 0.5% U-[13C] glucose (Campro Scientific). Bacteria were grown TR:TREATMENT_SUMMARY for 72h, washed with PBS and subjected to heat-killing by re-suspension in PBS TR:TREATMENT_SUMMARY and subsequently incubation at 60˚C for 60-90 min. Bacteria were kept at 4˚C TR:TREATMENT_SUMMARY until use. Efficient killing was confirmed by overnight plating on LB-agar TR:TREATMENT_SUMMARY plates. For DAPI labelling, killed bacteria were incubated with 0.2 ug/ml of TR:TREATMENT_SUMMARY DAPI in PBS for 5min and washed 3 times with cold 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 AA89-53_r97- AA89-54_r38- AA89-55_r110- AA89-56_r96- AA89-57_r41- AA89-58_r71- AA89-59_r64- AA89-60_r95- AA89-61_r19- AA89-62_r17- Factors treatment:control | Sample source:murine peritoneal macrophages treatment:control | Sample source:murine peritoneal macrophages treatment:control | Sample source:murine peritoneal macrophages treatment:control | Sample source:murine peritoneal macrophages treatment:control | Sample source:murine peritoneal macrophages treatment:Ecoli treated | Sample source:murine peritoneal macrophages treatment:Ecoli treated | Sample source:murine peritoneal macrophages treatment:Ecoli treated | Sample source:murine peritoneal macrophages treatment:Ecoli treated | Sample source:murine peritoneal macrophages treatment:Ecoli treated | Sample source:murine peritoneal macrophages ADP 188634 647786.81 763143.81 62501.99 44952.58 175981.95 88335.66 111548.13 82249.7 33210.02 AMP 8403688 3809156 8984455 1755949.88 2713079.75 16251023 8558429 8228822.5 9323810 623902 AMP 13C2 8664.0852 2227.3724 0 1977.346452 0 217075.7117 94835.7391 89702.36775 87734.069 3687.8258 AMP 13C3 18165.02 3843.15 12651.92 6145.77 3766.71 173252.27 98525.44 65832.6 81812.73 14439.88 AMP 13C4 13643.48 0 0 0 3527.81 91040.28 76345.96 53122.97 74482.2 5150.23 AMP 13C5 43446.77 13628.94 36753.46 33181.54 12215.86 492960.72 270912 172455.42 287036.09 29287.46 AMP 13C6 311578.97 521721.66 350142 395091.75 136622.83 267142.53 221361.88 256459.31 195728.83 7220.53 AMP 13C8 38867.61 43629.97 42314.8 9346.84 7322.37 111042.56 105718.54 111852.23 101346.9 17951.52 AMP 13C9 19190.13 6136.89 13048.82 8323.46 6593.32 174974.92 103879.05 81509.4 78696.1 6708.06 AMP 13C10 71052.27 46091.04 70873.59 11509.08 31543.54 804316.75 594221.19 374352.25 468048.91 63573.89 Adenosine 29966.01 29821.6 13982.01 9549.03 17578.24 117644.02 105194.01 54431.79 64017.51 33904.71 Adenosine 13C10 141386.3 116201.87 83130.19 137562.83 110599.95 118001.47 119163.41 119868.69 86023.6 103921.43 Adenine 524849.62 729296.81 962836.56 432636.66 823755.25 559256.69 416745.72 421383.16 262027.95 334801.12 Citrate 2487836.25 4123848 3201094.25 926299.69 989299.31 3026014.75 2554589.75 2154570.75 3456820.25 733530.69 Citrate 13C2 81368.8253 162090.6881 123730.5334 24197.80855 33492.40221 326371.2697 405733.367 266048.7798 395067.341 57341.46031 Citrate 13C3 0 16987.05 9819.77 0 0 68591.35 72465.6 45469.14 88363.29 3988.64 Citrate 13C4 83446.62 25184.95 46857.07 37771.56 57229.68 189209.73 271270.09 163394.73 307452.03 45096.15 Citrate 13C6 0 0 0 0 0 0 11059.4922 4831.9004 13233.4795 0 2-Oxoglutarate 130857.19 133090.36 160689.16 105486.5 44338.86 319127.81 181919.33 151261.88 91785.01 59692.12 Succinate 1061078.38 757086.69 799056 436365.72 579054.56 531946.25 494815.5 394430.03 584811.25 601592.56 Succinate 13C4 0 0 0 0 0 4802.2153 13195.0361 5677.6973 4109.0674 0 Fumarate 309603.78 719175.56 389455.16 243300.38 136789.25 955688.25 819693.31 629197.62 1361408.5 80564.51 Fumarate 13C2 0 5035.776116 4981.60481 1378.190464 10651.68601 31352.16755 34321.14375 29843.77341 15700.27314 0 Malate 4067637.25 5869827.5 3789770.75 2897682.75 1590986.38 5513214 4940396.5 5288491.5 6764582 1258994.62 Malate 13C2 117708.4043 183777.624 124444.9538 77922.6362 27360.51037 420544.3877 500077.5124 456761.2005 527194.6492 78782.04642 Malate 13C3 11222.1533 11836.8545 12977.8047 3242.1199 0 75329.2109 93103.8984 92918.375 115699.3516 15952.4297 Malate 13C4 40032.1094 111967.5547 84540.7109 41263.9883 4961.457 465159.6563 610180.3125 535241.75 649619.1875 172405.0469 Itaconate 679232.44 1046720.81 628542.19 333028.75 145709.83 1156660.75 768419.06 679415.5 572648.06 106621.63 Itaconate 13C2 16804.05187 25494.80955 0 7474.428031 3857.777764 73748.09123 38266.17234 43701.54811 19742.26962 1756.919569 2' 3'-Cyclic CMP 100263.5 113837.12 156857.92 15470.47 43990.06 52700 41163.33 99530.94 160073.39 27561.09 2' 3'-Cyclic CMP 13C9 50943.11 54285.57 64603.88 14475.95 49823.42 31383.83 9048.36 43552.03 11804.69 14045.12 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID med Mz med RT ADP C00008 426.022522 0.557 AMP C00020 346.055786 0.605 AMP 13C2 C00020 348.062836 0.617 AMP 13C3 C00020 349.069092 0.622 AMP 13C4 C00020 350.070343 0.612 AMP 13C5 C00020 351.071838 0.621 AMP 13C6 C00020 352.081818 0.589 AMP 13C8 C00020 354.073364 0.601 AMP 13C9 C00020 355.081573 0.606 AMP 13C10 C00020 356.089355 0.605 Adenosine C00121 266.089539 0.636 Adenosine 13C10 C00121 276.11734 0.636 Adenine C00147 134.047104 0.636 Citrate C00158 191.019577 0.543 Citrate 13C2 C00158 193.025787 0.54 Citrate 13C3 C00158 194.029938 0.544 Citrate 13C4 C00158 195.031006 0.556 Citrate 13C6 C00158 196.034406 0.556 2-Oxoglutarate C00026 145.014343 0.562 Succinate C00042 117.01947 0.559 Succinate 13C4 C00042 121.03307 0.559 Fumarate C00122 115.00383 0.559 Fumarate 13C2 C00122 117.010338 0.552 Malate C00149 133.014404 0.55 Malate 13C2 C00149 135.018967 0.552 Malate 13C3 C00149 136.022367 0.552 Malate 13C4 C00149 137.025767 0.552 Itaconate C00490 129.01947 0.568 Itaconate 13C2 C00490 131.025009 0.565 2' 3'-Cyclic CMP C02354 304.03418 0.584 2' 3'-Cyclic CMP 13C9 C02354 313.065094 0.525 METABOLITES_END #END