#METABOLOMICS WORKBENCH xu447338358_20240727_070416 DATATRACK_ID:5040 STUDY_ID:ST003389 ANALYSIS_ID:AN005558 PROJECT_ID:PR002100 VERSION 1 CREATED_ON August 8, 2024, 3:20 am #PROJECT PR:PROJECT_TITLE The metabolome analysis of energy metabolites after Salmonella Typhimurium PR:PROJECT_TITLE infection in mouse macrophages PR:PROJECT_TYPE The metabolome analysis of energy metabolites PR:PROJECT_SUMMARY The metabolome analysis of energy metabolites after Salmonella Typhimurium PR:PROJECT_SUMMARY infection in mouse macrophages. Glucose metabolites concentrations in S. PR:PROJECT_SUMMARY Typhimurium-infected (MOI = 10, 6 h) and Mock-infected mouse Peritoneal PR:PROJECT_SUMMARY macrophages. PMs were also pretreated with 2-DG (10 mM, 18 h) and infected with PR:PROJECT_SUMMARY S. Typhimurium (MOI =10, 6 h) or Mock-infected. PMs were also pretreated with PR:PROJECT_SUMMARY Rotenone (2.5 μM, 4 h) and infected with S. Typhimurium (MOI =10, 6 h) or PR:PROJECT_SUMMARY Mock-infected. PR:INSTITUTE Northwest A&F University PR:LAST_NAME Xu PR:FIRST_NAME Lei PR:ADDRESS Yangling, Xinong Road, Xianyang, 712100, China PR:EMAIL xulei@nwafu.edu.cn PR:PHONE 18149321513 #STUDY ST:STUDY_TITLE Disruption of Glucose Homeostasis by Bacterial Infection Orchestrates Host ST:STUDY_TITLE Innate Immunity Through NAD+/NADH Balance ST:STUDY_SUMMARY Metabolic reprogramming is crucial for activating innate immunity in ST:STUDY_SUMMARY macrophages, and the accumulation of immunometabolites is thought essential for ST:STUDY_SUMMARY effective defense against infection. The NAD+/NADH redox couple serves as a ST:STUDY_SUMMARY critical node that integrates metabolic pathways and signaling events, but how ST:STUDY_SUMMARY this metabolite couple engages macrophage activation remains unclear. Here, we ST:STUDY_SUMMARY showed that the NAD+/NADH ratio serves as a molecular signal that regulates ST:STUDY_SUMMARY proinflammatory responses and type I interferon (IFN) responses divergently. ST:STUDY_SUMMARY Salmonella Typhimurium infection led to a decreased NAD+/NADH ratio by inducing ST:STUDY_SUMMARY the accumulation of NADH. Further investigation showed that an increased ST:STUDY_SUMMARY NAD+/NADH ratio correlates to attenuated proinflammatory responses and enhanced ST:STUDY_SUMMARY type I IFN responses. Conversely, a decreased NAD+/NADH ratio is linked to ST:STUDY_SUMMARY intensified proinflammatory responses and restrained type I IFN responses. These ST:STUDY_SUMMARY results showed the NAD+/NADH ratio is an essential cell-intrinsic factor that ST:STUDY_SUMMARY orchestrates innate immunity, which enhanced our understanding of how ST:STUDY_SUMMARY metabolites fine-tune innate immunity. ST:INSTITUTE Northwest A&F University ST:LAST_NAME Xu ST:FIRST_NAME Lei ST:ADDRESS Yangling, Xinong Road, Xiyang, 712100, China ST:EMAIL xulei@nwafu.edu.cn ST:PHONE 18149321513 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #FACTORS #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 - STPM1 Sample source:Peritoneal macrophages | Factor:Control1 RAW_FILE_NAME(Raw file name)=CK1 SUBJECT_SAMPLE_FACTORS - STPM2 Sample source:Peritoneal macrophages | Factor:Control2 RAW_FILE_NAME(Raw file name)=CK1 SUBJECT_SAMPLE_FACTORS - STPM3 Sample source:Peritoneal macrophages | Factor:Control3 RAW_FILE_NAME(Raw file name)=CK3 SUBJECT_SAMPLE_FACTORS - STPM4 Sample source:Peritoneal macrophages | Factor:Salmonella Typhimurium infection1 RAW_FILE_NAME(Raw file name)=SL1 SUBJECT_SAMPLE_FACTORS - STPM5 Sample source:Peritoneal macrophages | Factor:Salmonella Typhimurium infection2 RAW_FILE_NAME(Raw file name)=SL2 SUBJECT_SAMPLE_FACTORS - STPM6 Sample source:Peritoneal macrophages | Factor:Salmonella Typhimurium infection3 RAW_FILE_NAME(Raw file name)=SL3 SUBJECT_SAMPLE_FACTORS - STPM7 Sample source:Peritoneal macrophages | Factor:2DG+Salmonella Typhimurium infection1 RAW_FILE_NAME(Raw file name)=2DG-SL2 SUBJECT_SAMPLE_FACTORS - STPM8 Sample source:Peritoneal macrophages | Factor:2DG+Salmonella Typhimurium infection2 RAW_FILE_NAME(Raw file name)=2DG-SL2 SUBJECT_SAMPLE_FACTORS - STPM9 Sample source:Peritoneal macrophages | Factor:2DG+Salmonella Typhimurium infection3 RAW_FILE_NAME(Raw file name)=2DG-SL3 SUBJECT_SAMPLE_FACTORS - STPM10 Sample source:Peritoneal macrophages | Factor:Rotenone+Salmonella Typhimurium infection1 RAW_FILE_NAME(Raw file name)=Re-SL1 SUBJECT_SAMPLE_FACTORS - STPM11 Sample source:Peritoneal macrophages | Factor:Rotenone+Salmonella Typhimurium infection2 RAW_FILE_NAME(Raw file name)=Re-SL2 SUBJECT_SAMPLE_FACTORS - STPM12 Sample source:Peritoneal macrophages | Factor:Rotenone+Salmonella Typhimurium infection3 RAW_FILE_NAME(Raw file name)=Re-SL3 #COLLECTION CO:COLLECTION_SUMMARY Cells in plates were counted, washed with cold PBS and then flash-frozen in CO:COLLECTION_SUMMARY liquid nitrogen CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY The metabolome analysis of energy metabolites after Salmonella Typhimurium TR:TREATMENT_SUMMARY infection in mouse macrophages. Glucose metabolites concentrations in S. TR:TREATMENT_SUMMARY Typhimurium-infected (MOI = 10, 6 h) and Mock-infected mouse Peritoneal TR:TREATMENT_SUMMARY macrophages. PMs were also pretreated with 2-DG (10 mM, 18 h) and infected with TR:TREATMENT_SUMMARY S. Typhimurium (MOI =10, 6 h) or Mock-infected. PMs were also pretreated with TR:TREATMENT_SUMMARY Rotenone (2.5 μM, 4 h) and infected with S. Typhimurium (MOI =10, 6 h) or TR:TREATMENT_SUMMARY Mock-infected. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 2 × 107 cells were harvested and thawed at 4 °C mixed with 200 μL water, and SP:SAMPLEPREP_SUMMARY 800 μL of cold methanol/acetonitrile (1:1, v/v) to remove the protein. The SP:SAMPLEPREP_SUMMARY mixture was centrifuged for 15 min (14000 × g, 4°C). The supernatant was dried SP:SAMPLEPREP_SUMMARY in a vacuum centrifuge. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 15 mM ammonium acetate CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT 0-18 min: 90% B to 40% B, 18-18.1 min: 40% B to 90% B, 18.1-23 min: 90% B. CH:FLOW_RATE 300 μL/min CH:COLUMN_TEMPERATURE 35 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS sheath gas temperature: 350°C; ion Source Gas1 (Gas1): 45 psi, Ion Source Gas2 MS:MS_COMMENTS (Gas2): 45 psi, Curtain gas (CUR): 30 psi; ionSapary Voltage Floating (ISVF): MS:MS_COMMENTS 4500 V; nebulizer pressure: 40psi. The mass spectrometer operated with a dwell MS:MS_COMMENTS time of 200 ms. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS μmol/g MS_METABOLITE_DATA_START Samples STPM1 STPM2 STPM3 STPM4 STPM5 STPM6 STPM7 STPM8 STPM9 STPM10 STPM11 STPM12 Factors Sample source:Peritoneal macrophages | Factor:Control1 Sample source:Peritoneal macrophages | Factor:Control2 Sample source:Peritoneal macrophages | Factor:Control3 Sample source:Peritoneal macrophages | Factor:Salmonella Typhimurium infection1 Sample source:Peritoneal macrophages | Factor:Salmonella Typhimurium infection2 Sample source:Peritoneal macrophages | Factor:Salmonella Typhimurium infection3 Sample source:Peritoneal macrophages | Factor:2DG+Salmonella Typhimurium infection1 Sample source:Peritoneal macrophages | Factor:2DG+Salmonella Typhimurium infection2 Sample source:Peritoneal macrophages | Factor:2DG+Salmonella Typhimurium infection3 Sample source:Peritoneal macrophages | Factor:Rotenone+Salmonella Typhimurium infection1 Sample source:Peritoneal macrophages | Factor:Rotenone+Salmonella Typhimurium infection2 Sample source:Peritoneal macrophages | Factor:Rotenone+Salmonella Typhimurium infection3 Succinate 0.977563133 0.773046231 0.84737383 0.953232702 1.160490706 1.039085802 1.374222072 1.35532705 1.283232127 1.8179206 1.752599396 1.661659943 Phosphoenolpyruvate 0.119423124 0.112919813 0.139899829 0.124742268 0.149543577 0.137209627 0.230823148 0.204063494 0.201506946 0.252979033 0.258513786 0.231272244 L-Malic acid 2.795022671 2.502031499 2.405961572 3.154865346 2.622467487 3.233774074 1.192374305 1.608060614 2.281753517 0.926074213 0.907269507 0.646002475 Lactate 7.227226505 8.936543182 9.688132759 13.18115406 9.912720841 14.50497113 7.472968509 11.63942334 9.695499638 16.09845421 8.457283918 9.732133244 cis-Aconitate 0.050309937 0.042695302 0.031050351 0.029697452 0.044019073 0.056081861 0.042115466 0.034298158 0.026327658 0.022756051 0.021772774 0.030008001 GMP 0.118248329 0.176985151 0.134994141 0.085749924 0.090777284 0.105202941 0.207576349 0.106476308 0.061977645 0.077548417 0.101370352 0.063662451 NAD 0.004256094 0.004230355 0.005062728 0.003678152 0.003791714 0.004667517 0.004382657 0.004016247 0.003829267 ADP 0.073272674 0.042007903 0.048655568 0.054546867 0.066240607 0.061388308 0.042625937 0.047800569 0.051491547 0.05840224 0.049822453 0.035445409 Adenosine monophosphate 0.061637913 0.061796036 0.047125261 0.043685645 0.056030547 0.05764616 0.083038289 0.043833671 0.042823152 0.044642665 0.044229395 0.036717001 Cyclic AMP 0.014738621 0.012290582 0.013287684 0.013839339 0.013688181 0.01354063 0.017206884 0.017265675 0.015895395 0.021632676 0.021649182 0.020931323 Isocitrate 0.547057018 0.463944577 0.501724195 0.529753852 0.624983933 0.588497998 0.70851054 0.770340176 0.712329889 0.977723369 0.952450987 0.904609249 Citrate 1.250426893 0.996308811 0.991245253 0.842143166 1.143675006 1.199159814 1.834089963 0.829514063 0.83872073 0.843449395 0.78850665 0.828563012 Fumarate 0.310077016 0.424448954 0.25634366 0.663625313 0.452736494 0.719252673 0.146476469 0.42363579 0.283597449 0.368115813 0.307711628 0.083351157 Guanosine 5'-diphosphate (GDP) 0.090677283 0.07077861 0.077979825 0.078412419 0.097798905 0.08583778 0.109092881 0.11648481 0.114281328 0.148943573 0.146440035 0.136769487 Thiamine pyrophosphate (TPP) 0.032402013 0.025861936 0.027531355 0.02972113 0.03055904 0.02553918 0.034614118 0.032624771 0.031955208 0.047027753 0.043400522 0.040926109 Flavin mononucleotide (FMN) 0.026884877 0.022477544 0.028455494 0.021619808 0.027236033 0.024893947 0.033318733 0.030560254 0.036110701 0.032560087 0.033832611 D-Fructose 1,6-bisphosphate 0.861360445 0.694685638 0.789515307 0.875674248 1.025340464 0.951059762 1.131936166 1.252228842 1.233106517 1.649477375 1.612166777 1.514519481 Beta-D-Fructose 6-phosphate 1.984206044 2.94631403 2.333669606 2.367419409 2.109908955 2.835178205 0.761701625 1.076139658 1.394511258 0.901919644 1.203295642 0.244627597 3-Phospho-D-glycerate 0.071794554 0.082387551 0.207242826 0.050307081 0.04472539 0.058834869 0.177329401 0.17456247 0.220100841 0.078083977 0.135408001 0.052658803 D-Glucose 6-phosphate 4.454401141 5.977842039 4.80298369 4.66917726 4.606835197 5.284612582 1.439079448 2.111433598 2.840369633 1.45052211 2.359613583 0.236502828 Dihydroxyacetone phosphate 1.583603856 3.326178666 2.217814877 3.792259831 3.233899654 3.655356033 2.810008672 3.788368013 3.535684827 2.950125662 3.31755694 2.090557769 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID Transitions Retention Time(min) QC RSD Succinate C00042 117.1 / 73.0 4.673588078 0.003442409 Phosphoenolpyruvate C00074 167.1 / 79.0 7.630280642 0.012638918 L-Malic acid C00149 133.0 / 115.1 5.114656026 0.031495835 Lactate C00186 89.2 / 43.0 1.75271006 0.016738912 cis-Aconitate C00417 173.1 / 85.1 6.994462743 0.049707047 GMP C00144 362.1 / 79.1 7.820539849 0.038344069 NAD C00003 662.1 / 540.0 6.413869011 0.056172477 ADP C00008 426.0 / 79.0 7.546516319 0.043560178 Adenosine monophosphate C00020 346.1 / 79.0 6.200016303 0.011480671 Cyclic AMP C00575 328.0 / 134.0 1.940149594 0.031161813 Isocitrate C00311 191.0 / 73.0 7.779570647 0.01491163 Citrate C00158 191.1 / 87.1 7.851614054 0.038247269 Fumarate C00122 115.1 / 71.1 4.170084346 0.012580334 Guanosine 5'-diphosphate (GDP) C00035 442.0 / 79.0 9.173402084 0.010170496 Thiamine pyrophosphate (TPP) C00068 423.0 / 302.0 9.156052504 0.032987736 Flavin mononucleotide (FMN) C00061 455.1 / 97.0 5.13836164 0.047847219 D-Fructose 1,6-bisphosphate C00354 339.1 / 97.0 11.12847048 0.008223766 Beta-D-Fructose 6-phosphate C05345 259.1 / 97.0 7.357142666 0.089065012 3-Phospho-D-glycerate C00197 185.0 / 97.0 7.939306553 0.100665105 D-Glucose 6-phosphate C00092 259.0 / 97.0 8.330382053 0.025603914 Dihydroxyacetone phosphate C00111 169.1 / 97.0 6.226807543 0.076317946 METABOLITES_END #END