{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003607","ANALYSIS_ID":"AN005927","PROJECT_ID":"PR002231","VERSION":"1","CREATED_ON":"December 5, 2024, 7:47 pm"}, "PROJECT":{"PROJECT_TITLE":"Macrophages recycle phagocytosed bacteria to fuel immunometabolic responses","PROJECT_SUMMARY":"Macrophages specialize in phagocytosis, a cellular process that eliminates extracellular matter, including microbes, through internalization and degradation. Despite the critical role of phagocytosis during bacterial infection, the fate of phagocytosed microbial cargo and its impact on host cell is poorly understood. Here, we reveal that ingested bacteria constitute an alternative nutrient source that skews immunometabolic host responses. Tracing stable isotope-labelled bacteria, we found that phagolysosomal degradation of bacteria provides carbon atoms and amino acids that are recycled into various metabolic pathways, including glutathione and itaconate biosynthesis, and satisfy macrophage bioenergetic needs. Metabolic recycling of microbially-derived nutrients is regulated by the nutrient sensing mTORC1 and intricately tied to microbial viability. Dead bacteria, as opposed to live ones, are enriched in cyclic- adenosine monophosphate (AMP), sustain the cellular AMP pool and subsequently activate AMP protein kinase (AMPK) to inhibit mTORC1. Consequently, killed bacteria strongly fuel metabolic recycling and support macrophage survival, but elicit decreased reactive oxygen species (ROS) production and a reduced IL-1β secretion compared to viable bacteria. These results reveal a novel insight into the fate of engulfed microbes and highlights a microbial viability-associated metabolite that triggers host metabolic and immune responses. Our findings hold promise for shaping immunometabolic intervention in various immune-related pathologies.","INSTITUTE":"University of Colorado Anschutz Medical Campus","LABORATORY":"Lab of Angelo D''Alessandro in collaboration with lab of Johan Garaude (INSERM, Fr)","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"}, "STUDY":{"STUDY_TITLE":"Global metabolomics and tracing of 13C-labeled metabolites of bone marrow derived macrophages following engulfment of dead 13C-labeled Citrobacter rodentium, Listeria innocua, Staphilococcus aureus, or Pseudomonas aeruginosa.","STUDY_SUMMARY":"Global metabolomics and 13C-tracing analysis of bone marrow derived macrophages exposed to heat-killed uniformly 13C-labelled Citrobacter rodentium, Listeria innocua, Staphilococcus aureus or Pseudomonas aeruginosa for 4h.","INSTITUTE":"University of Colorado Anschutz Medical Campus","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"}, "SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","GENDER":"Not applicable"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"-", "Sample ID":"AA89-33_r67+", "Factors":{"treatment":"CTRL","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-33_r67"} }, { "Subject ID":"-", "Sample ID":"AA89-34_r102+", "Factors":{"treatment":"CTRL","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-34_r102"} }, { "Subject ID":"-", "Sample ID":"AA89-35_r39+", "Factors":{"treatment":"CTRL","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-35_r39"} }, { "Subject ID":"-", "Sample ID":"AA89-36_r15+", "Factors":{"treatment":"CTRL","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-36_r15"} }, { "Subject ID":"-", "Sample ID":"AA89-37_r55+", "Factors":{"treatment":"Killed-Citro","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-37_r55"} }, { "Subject ID":"-", "Sample ID":"AA89-38_r8+", "Factors":{"treatment":"Killed-Citro","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-38_r8"} }, { "Subject ID":"-", "Sample ID":"AA89-39_r42+", "Factors":{"treatment":"Killed-Citro","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-39_r42"} }, { "Subject ID":"-", "Sample ID":"AA89-40_r32+", "Factors":{"treatment":"Killed-Citro","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-40_r32"} }, { "Subject ID":"-", "Sample ID":"AA89-41_r53+", "Factors":{"treatment":"Killed-Listeria","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-41_r53"} }, { "Subject ID":"-", "Sample ID":"AA89-42_r38+", "Factors":{"treatment":"Killed-Listeria","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-42_r38"} }, { "Subject ID":"-", "Sample ID":"AA89-43_r65+", "Factors":{"treatment":"Killed-Listeria","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-43_r65"} }, { "Subject ID":"-", "Sample ID":"AA89-44_r43+", "Factors":{"treatment":"Killed-Listeria","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-44_r43"} }, { "Subject ID":"-", "Sample ID":"AA89-45_r105+", "Factors":{"treatment":"Killed_Staph","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-45_r105"} }, { "Subject ID":"-", "Sample ID":"AA89-46_r70+", "Factors":{"treatment":"Killed_Staph","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-46_r70"} }, { "Subject ID":"-", "Sample ID":"AA89-47_r81+", "Factors":{"treatment":"Killed_Staph","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-47_r81"} }, { "Subject ID":"-", "Sample ID":"AA89-48_r80+", "Factors":{"treatment":"Killed_Staph","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-48_r80"} }, { "Subject ID":"-", "Sample ID":"AA89-49_r108+", "Factors":{"treatment":"Killed-Pseudo","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-49_r108"} }, { "Subject ID":"-", "Sample ID":"AA89-50_r36+", "Factors":{"treatment":"Killed-Pseudo","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-50_r36"} }, { "Subject ID":"-", "Sample ID":"AA89-51_r33+", "Factors":{"treatment":"Killed-Pseudo","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-51_r33"} }, { "Subject ID":"-", "Sample ID":"AA89-52_r12+", "Factors":{"treatment":"Killed-Pseudo","Sample source":"murine bone marrow derived macrophages"}, "Additional sample data":{"RAW_FILE_NAME(raw file name)":"AA89-52_r12"} } ], "COLLECTION":{"COLLECTION_SUMMARY":"BMDMs were seeded at 2.5E6 cell/well in a non-tissue treated 6-well plate (2 wells per conditions) the day before the experiment. For metabolite extraction, BMDMs were washed with cold PBS and harvested with 5 mM EDTA in PBS, frozen as dry cell pellet, and stored at -80˚C until processing.","SAMPLE_TYPE":"Macrophages"}, "TREATMENT":{"TREATMENT_SUMMARY":"Preparation of macrophages: Murine bone marrow-derived macrophages (BMDMs) were generated in RPMI 1640 supplemented with M-CSF (30% mycoplasma-free L929 cell supernatant, NCBI Biosample accession # SAMN00155972) and 10% FBS, plus 100 µg/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 1 nM sodium pyruvate and 50 µM 2-mercaptoethanol (all from Gibco). BMDMs were used on day 5 to 7 after seeding. Period of differentiation of the cells, concentration of cells when replating and time-lapse between replating and stimulation with bacteria were important parameters to maintain metabolic backgrounds homogenous between experiments. Preparation of viable and killed U-[13C]Bacteria: Citrobacter rodentium, Staphylococus aureus, Listeria innocua, Pseudomonas aeruginosa were grown overnight with shaking in LB, diluted 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with phosphate buffer saline (PBS) to remove LB salts before addition to cells. For labeling, 10 µl of an overnight cultured of bacteria was added to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, and 0.5% U-13C glucose (Campro Scientific). Bacteria were grown for 72h, washed with PBS and subjected to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. For antibiotic killing, bacteria were incubated for 6h to 12h with streptomycin or gentamycin (50 µg/ml). Bacteria were kept at 4˚C until use. Efficient killing was confirmed by overnight plating on LB-agar plates. Treatment of macrophages: 2E6 BMDMs were plated 12-16h prior stimulation in non-tissue cultured treated 6-well plate (BD Falcons). Cells were then stimulated with killed labelled bacteria at MOI 25, centrifuged at 2000 rpm for 5 min. BMDMs were incubated for 5 min and washed with PBS to remove non-ingested bacteria and further incubated for 4h."}, "SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Metabolites from frozen pellets were extracted at 4e6 cells per mL using ice cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 degrees C followed by centrifugation as described for 10 min at 18,000 g. Supernatants were maintained at 4°C until analysis that same day.","PROCESSING_STORAGE_CONDITIONS":"4℃","EXTRACT_STORAGE":"-80℃"}, "CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Negative C18","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)","SOLVENT_A":"95% water/5% acetonitrile; 1 mM ammonium acetate","SOLVENT_B":"95% acetonitrile/5% water; 1 mM ammonium acetate","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 100-0% B, 3-5 min hold at 0% B","FLOW_RATE":"450 uL/min","COLUMN_TEMPERATURE":"45","SAMPLE_INJECTION":"10 uL"}, "ANALYSIS":{"ANALYSIS_TYPE":"MS"}, "MS":{"INSTRUMENT_NAME":"Thermo Orbitrap Exploris 120","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) detection duration 20 msec, source voltage 2.0 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database."}, "MS_METABOLITE_DATA":{ "Units":"peak area", 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