#METABOLOMICS WORKBENCH juliehaines_20241126_074650 DATATRACK_ID:5406 STUDY_ID:ST003611 ANALYSIS_ID:AN005936 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 Positive 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 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min CH:FLOW_GRADIENT 95-5% B, 3-5 min hold at 5% 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 POSITIVE 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 3.5 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 Arginine 1690211.12 1316758.12 2245306.25 1230951.38 2332888 2552984 2717759.5 2440210.25 3192272 1626159 Arginine 13C3 9178.18 22920.18 17375.52 4625.99 12829.52 14959.83 45047.7 6382.36 22087.53 3522.91 Arginine 13C4 40902.46 26869.82 42582.79 19553.53 4752 46823.21 69994.35 50976.97 67375.6 8958.13 Arginine 13C5 20433.32 14813.65 13789.06 6915.83 6108.13 79678.87 114067.78 48287.32 87642.19 30804.21 Arginine 13C6 74762.8 51660.14 42960.81 51056.86 23975.32 346067.03 416019.75 226021.42 387703.12 121969.8 Cysteine 494396.88 532368.5 478710.75 434952.09 43268.91 5302613.5 5075747.5 4251465.5 4520884.5 240336.3 Cysteine 13C3 35572.95 55451.17 31507.13 30811.81 0 487189.03 642604.5 396178.5 435980.09 59762.31 Glutamate 2180610 2758086.75 2214423.25 2189113 930663.75 6688388.5 5407099.5 4831601 5419846 671468.81 Glutamate 13C2 50333.45475 101532.2776 65285.98967 64409.27318 19605.12216 529900.9348 559386.774 367996.817 376467.3059 34903.56685 Glutamate 13C3 44646.68 53553.15 34624.68 35230.19 3503.84 805217.19 890041 574900.44 686741.81 86674.23 Glutamate 13C4 15560.91 28013.38 28902.1 34886.39 33427.73 93592.79 121208.24 83455.84 90474.99 20390.4 Glutamate 13C5 34838.05 52450.41 26132.44 30901.24 8685.58 636208.19 801759 506280.28 642192.75 103252.62 Glutamine 826225.31 810039.5 936286.75 885940.94 650395.25 3056562.25 2878396 2659265.25 2756883.75 721872.94 Glutamine 13C2 1648.348437 2959.000513 0 0 1.04436875 19154.68919 24101.0221 9687.002619 11819.83666 4786.234357 Glutamine 13C3 0 3517.52 3159 0 0 47453.96 46092.58 42427.07 41481.95 4320.37 Glutamine 13C5 12509.8 25117.94 11762.64 23752.03 0 187426.8 216054.88 134564.95 156751.05 23494.63 Methionine 336734.53 263966.84 338179.16 167332.31 131469.42 1972400.12 1972531 2324051.75 2268267.25 91253.88 Methionine 13C2 5100.398047 8060.270309 19171.04804 0 5317.835005 17043.92964 27541.98373 13442.32346 12134.31157 4396.057013 Methionine 13C3 0 0 0 0 0 8787.2793 12737.5576 18217.1504 11478.4971 1490.2167 Methionine 13C4 0 1954.9775 7415.2065 0 0 46626.4844 58953.9063 36166.3867 48105.8906 4931.8086 Methionine 13C5 55632.64 61059.92 61383.08 37973.5 27678.33 516706.88 368815.97 427854.59 353801.41 71184.51 Glutathione 14421756 25209776 15970541 10276800 6195178 19495278 12692864 10287968 13700764 564936.94 Glutathione 13C2 0 0 0 0 0 837414.5162 714646.7856 454129.8972 599577.2556 28454.52303 Glutathione 13C3 41886.85 56708.82 26480.91 22261.54 9700.37 557557 392930.09 243077.23 334042.28 18488.52 Glutathione 13C4 0 0 6710.81 2487.97 0 169777.31 140832.56 83462.69 134507.95 7131.78 Glutathione 13C5 23320.75 41919.01 8092.18 6710.87 0 415747 430341.22 265819.94 351348 21836.54 Glutathione 13C6 28272.61 28172.59 49348.97 18807.59 8593.19 190716.19 102155.41 71927.86 103695.97 20329.99 Glutathione 13C7 0 0 0 0 0 146413.9219 156438.4219 103430.7813 109912.0391 9748.8945 Glutathione 13C8 4115.16 2256.1 0 0 0 57779.23 39972.65 26527.05 40670.62 7374.98 Glutathione 13C10 5535.71 0 4920.79 0 2635.37 9019.32 25456.28 14396.33 10234.46 1701.72 Glutathione disulfide 13C2 40874.69744 89141.81196 52513.58247 28139.17 0 48336.38 16280.45 28215.35 30685.26 0 Glutathione disulfide 13C3 5614.98 34558.09 8209.48 4255.85 0 0 6474.55 8146 1329.02 0 Cystathionine 36082.11 32049.98 32065.85 25266.81 2426.49 96216.34 86278.38 59275.86 81007.62 1531.83 Cystathionine 13C7 935337.81 2635984.25 1834929.38 1635479 152909.11 310004.34 362531.53 512287.47 410021.91 19468.27 S-Adenosyl-L-methionine 8981.75 9999.29 15222.87 18453.47 0 58301.84 47968.14 46387.66 38397.13 0 Putrescine 101156.55 111771.25 128721.31 107357.65 80369.74 274275.12 206232.73 152800.05 178092.3 31305.3 Putrescine 13C4 0 4154.65 0 3552.19 0 102885.27 105442.88 44410.91 67141.54 20680.96 Spermidine 860601.75 777178.5 129422.06 375069.59 448777.16 127924.84 469815.75 87080.88 234486.11 7548.93 Spermine 39765.95 48763.13 10403.33 37363.02 77948.73 5178.67 15839.68 0 37354.27 0 Ornithine 425238.78 453047.84 453086.75 243554 203065.08 3506709.25 3183583 2948283.25 2615886.25 439333.16 Ornithine 13C2 0 0 686.8678813 0 0 30493.09122 29152.80593 4907.508569 1101.396194 10171.89999 Ornithine 13C3 1764.3 17333.81 0 6709.37 0 236358.81 240460.98 174506.36 164771.08 62273.31 Ornithine 13C4 0 0 0 0 0 102965.37 114768.47 89401.75 71948.22 11264.94 Ornithine 13C5 48354.52 32925.05 17503.52 32918.14 4416.1 877392.19 1009395.5 650138.06 652201.31 197033.5 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID med Mz med RT Arginine C00062 175.119095 0.586 Arginine 13C3 C00062 178.13092 0.617 Arginine 13C4 C00062 179.128723 0.628 Arginine 13C5 C00062 180.136261 0.618 Arginine 13C6 C00062 181.138931 0.591 Cysteine C00097 122.027283 0.626 Cysteine 13C3 C00097 125.037071 0.652 Glutamate C00025 148.060532 0.615 Glutamate 13C2 C00025 150.066727 0.635 Glutamate 13C3 C00025 151.070984 0.671 Glutamate 13C4 C00025 152.070648 0.641 Glutamate 13C5 C00025 153.076691 0.665 Glutamine C00064 147.076324 0.612 Glutamine 13C2 C00064 148.079724 0.612 Glutamine 13C3 C00064 150.086731 0.648 Glutamine 13C5 C00064 152.093201 0.619 Methionine C00073 150.058502 0.636 Methionine 13C2 C00073 152.068008 0.614 Methionine 13C3 C00073 153.071408 0.614 Methionine 13C4 C00073 154.074808 0.614 Methionine 13C5 C00073 155.073517 0.654 Glutathione C00051 308.091187 0.637 Glutathione 13C2 C00051 310.097107 0.644 Glutathione 13C3 C00051 311.100952 0.654 Glutathione 13C4 C00051 312.1026 0.661 Glutathione 13C5 C00051 313.107147 0.642 Glutathione 13C6 C00051 314.105286 0.573 Glutathione 13C7 C00051 315.108686 0.573 Glutathione 13C8 C00051 316.121887 0.651 Glutathione 13C10 C00051 318.125397 0.646 Glutathione disulfide 13C2 C00127 615.157227 0.634 Glutathione disulfide 13C3 C00127 616.168091 0.66 Cystathionine C00542 223.074493 0.637 Cystathionine 13C7 C00542 230.09758 0.626 S-Adenosyl-L-methionine C00019 399.144501 0.601 Putrescine C00134 89.107437 0.576 Putrescine 13C4 C00134 93.120972 0.573 Spermidine C00315 146.165176 0.567 Spermine C00750 203.222824 0.559 Ornithine C01602 133.097427 0.575 Ornithine 13C2 C01602 135.104227 0.575 Ornithine 13C3 C01602 136.10611 0.627 Ornithine 13C4 C01602 137.110641 0.624 Ornithine 13C5 C01602 138.114014 0.605 METABOLITES_END #END