#METABOLOMICS WORKBENCH Tixiao_20241010_184447 DATATRACK_ID:5282 STUDY_ID:ST003536 ANALYSIS_ID:AN005808 PROJECT_ID:PR002176 VERSION 1 CREATED_ON October 24, 2024, 4:53 pm #PROJECT PR:PROJECT_TITLE Metabolomics data of feces from Control and BLE mice PR:PROJECT_SUMMARY Untargeted metabolomic analysis showed that 38 metabolites were significantly PR:PROJECT_SUMMARY different between control and BLE mice. Among them, oligosaccharides (1-kestose, PR:PROJECT_SUMMARY raffinose, maltotriose), amino acids (N-Acetyl-L-leucine, glutamic acid) and PR:PROJECT_SUMMARY polyamine (spermidine, SPD) were the most varied enriched metabolites. PR:INSTITUTE Shandong University PR:LAST_NAME Wang PR:FIRST_NAME Tixiao PR:ADDRESS 44# Wenhua Xi Road, Jinan, Shandong Province, 250012, China PR:EMAIL tixwang@163.com PR:PHONE 18560082747 #STUDY ST:STUDY_TITLE Probiotics and their metabolite spermidine enhance IFN-γ+CD4+ T cell immunity ST:STUDY_TITLE to inhibit hepatitis B virus ST:STUDY_SUMMARY The therapeutic potential of commensal microbes and their metabolites is ST:STUDY_SUMMARY promising in the functional cure of chronic hepatitis B virus (HBV) infection, ST:STUDY_SUMMARY which is defined as HBsAg loss. Here, using both specific-pathogen-free and ST:STUDY_SUMMARY germ-free mice, we report that probiotics significantly promote the decline of ST:STUDY_SUMMARY HBsAg and inhibit HBV replication by enhancing intestinal homeostasis and ST:STUDY_SUMMARY provoking intrahepatic IFN-γ+CD4+ T cell immune response. Depletion of CD4+ T ST:STUDY_SUMMARY cells or blockage of IFN-γ abolishes probiotics-mediated HBV inhibition. ST:STUDY_SUMMARY Specifically, probiotics-derived spermidine accumulates in gut and transports to ST:STUDY_SUMMARY liver, where it exhibits a similar anti-HBV effect. Mechanistically, spermidine ST:STUDY_SUMMARY enhances IFN-γ+CD4+ T cell immunity by autophagy. Strikingly, administration of ST:STUDY_SUMMARY probiotics in HBV patients reveals a preliminary trend to accelerate the decline ST:STUDY_SUMMARY of serum HBsAg. In conclusion, probiotics and theirs derived spermidine promote ST:STUDY_SUMMARY HBV clearance via autophagy-enhanced IFN-γ+CD4+ T cell immunity, highlighting ST:STUDY_SUMMARY the therapeutic potential of probiotics and spermidine for the functional cure ST:STUDY_SUMMARY of HBV patients. ST:INSTITUTE Shandong University ST:LAST_NAME Wang ST:FIRST_NAME Tixiao ST:ADDRESS 44 Wenhua Xi Road, Jinan, Shandong ST:EMAIL tixwang@163.com ST:PHONE 18560082747 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male #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 - C2 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C2_1.cdf SUBJECT_SAMPLE_FACTORS - C3 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C3_1.cdf SUBJECT_SAMPLE_FACTORS - C4 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C4_1.cdf SUBJECT_SAMPLE_FACTORS - C5 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C5_1.cdf SUBJECT_SAMPLE_FACTORS - C6 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C6_1.cdf SUBJECT_SAMPLE_FACTORS - C7 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C7_1.cdf SUBJECT_SAMPLE_FACTORS - C8 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C8_1.cdf SUBJECT_SAMPLE_FACTORS - C9 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C9_1.cdf SUBJECT_SAMPLE_FACTORS - C10 Sample source:Feces | Treatment:Control RAW_FILE_NAME(Raw file name)=C10_1.cdf SUBJECT_SAMPLE_FACTORS - B1 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B1_1.cdf SUBJECT_SAMPLE_FACTORS - B2 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B2_1.cdf SUBJECT_SAMPLE_FACTORS - B4 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B4_1.cdf SUBJECT_SAMPLE_FACTORS - B5 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B5_1.cdf SUBJECT_SAMPLE_FACTORS - B6 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B6_1.cdf SUBJECT_SAMPLE_FACTORS - B7 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B7_1.cdf SUBJECT_SAMPLE_FACTORS - B8 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B8_1.cdf SUBJECT_SAMPLE_FACTORS - B9 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B9_1.cdf SUBJECT_SAMPLE_FACTORS - B10 Sample source:Feces | Treatment:BLE RAW_FILE_NAME(Raw file name)=B10_1.cdf #COLLECTION CO:COLLECTION_SUMMARY Male five-week-old C57BL/6J mice were hydrodynamically injected with 6 μg of CO:COLLECTION_SUMMARY AAV/HBV1.2 plasmid and gavage fed with probiotics (BLE, PBS as control) daily CO:COLLECTION_SUMMARY starting at 3 day post of hydrodynamically injection. Feces collected from CO:COLLECTION_SUMMARY Control and BLE mice after 6 weeks. CO:SAMPLE_TYPE Feces #TREATMENT TR:TREATMENT_SUMMARY Male five-week-old C57BL/6J mice were hydrodynamically injected with 6 μg of TR:TREATMENT_SUMMARY AAV/HBV1.2 plasmid and gavage fed with probiotics (BLEl) daily at 3 dpi of TR:TREATMENT_SUMMARY hydrodynamically injection. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Take 50±1mg sample into the 2mL EP tubes, extracted with 0.3mL extraction SP:SAMPLEPREP_SUMMARY liquid (VMethanol: VChlorofrom = 3:1), add 20μL of L-2-Chlorophenylalanine SP:SAMPLEPREP_SUMMARY (1mg/mL stock in dH2O) as internal standard, vortex mixing for 30s; Homogenized SP:SAMPLEPREP_SUMMARY in ball mill for 4min at 45Hz, then ultrasound treated for 5min (incubated in SP:SAMPLEPREP_SUMMARY ice water); Centrifuge for 15min at 12000rpm, 4℃; Transfer the supernatant SP:SAMPLEPREP_SUMMARY (0.2mL) into a fresh 2mL GC/MS glass vial , take 30μL from each sample and SP:SAMPLEPREP_SUMMARY pooling as QC sample. Dry completely in a vacuum concentrator without heating; SP:SAMPLEPREP_SUMMARY Add 30μL Methoxy amination hydrochloride (20mg/mL in pyridine) incubated for SP:SAMPLEPREP_SUMMARY 30min at 80℃; Add 40μL of the BSTFA regent (1% TMCS, v/v) to the sample SP:SAMPLEPREP_SUMMARY aliquots, incubated for 1.5h at 70℃; Add 5μL FAMEs (Standard mixture of fatty SP:SAMPLEPREP_SUMMARY acid methyl esters, C8-C16:1mg/mL; C18-C24:0.5mg/mL in chloroform) to the QC SP:SAMPLEPREP_SUMMARY sample when cooling to the room temperature; All samples were analyzed by gas SP:SAMPLEPREP_SUMMARY chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer SP:SAMPLEPREP_SUMMARY (GC-TOF-MS). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph system CH:CHROMATOGRAPHY_SUMMARY coupled with a Pegasus HT time-of-flight mass spectrometer. The system utilized CH:CHROMATOGRAPHY_SUMMARY a DB-5MS capillary column coated with 5% diphenyl cross-linked with 95% CH:CHROMATOGRAPHY_SUMMARY dimethylpolysiloxane (30m×250μm inner diameter, 0.25μm film thickness; J&W CH:CHROMATOGRAPHY_SUMMARY Scientific, Folsom, CA, USA). A 1μL aliquot of the analyte was injected in CH:CHROMATOGRAPHY_SUMMARY splitless mode. Helium was used as the carrier gas, the front inlet purge flow CH:CHROMATOGRAPHY_SUMMARY was 3mL min−1, and the gas flow rate through the column was 1mL min−1. The CH:CHROMATOGRAPHY_SUMMARY initial temperature was kept at 50°C for 1min, then raised to 310°C at a rate CH:CHROMATOGRAPHY_SUMMARY of 10°C min−1, then kept for 8min at 310°C. The injection, transfer line, CH:CHROMATOGRAPHY_SUMMARY and ion source temperatures were 280, 280, and 250°C, respectively. The energy CH:CHROMATOGRAPHY_SUMMARY was -70eV in electron impact mode. The mass spectrometry data were acquired in CH:CHROMATOGRAPHY_SUMMARY full-scan mode with the m/z range of 50-500 at a rate of 20 spectra per second CH:CHROMATOGRAPHY_SUMMARY after a solvent delay of 6.27min. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE - CH:COLUMN_TEMPERATURE 50°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7890B MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Chroma TOF 4.3X software of LECO Corporation and LECO-Fiehn Rtx5 database were MS:MS_COMMENTS used for raw peaks exacting, the data baselines filtering and calibration of the MS:MS_COMMENTS baseline, peak alignment, deconvolution analysis, peak identification and MS:MS_COMMENTS integration of the peak area1. Both of mass spectrum match and retention index MS:MS_COMMENTS match were considered in metabolites identification. Remove peaks detected in MS:MS_COMMENTS <50% of QC samples or RSD＞30% in QC samples. MS:MS_RESULTS_FILE ST003536_AN005808_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END