#METABOLOMICS WORKBENCH Peter_RS_20250813_033251 DATATRACK_ID:6277 STUDY_ID:ST004113 ANALYSIS_ID:AN006820 PROJECT_ID:PR002586 VERSION 1 CREATED_ON August 13, 2025, 2:29 pm #PROJECT PR:PROJECT_TITLE Taurine transport is a critical modulator of ionic fluxes during NLRP3 PR:PROJECT_TITLE inflammasome activation PR:PROJECT_TYPE MS exploratory analysis PR:PROJECT_SUMMARY Metabolic regulation is a key feature of inflammasome activation and effector PR:PROJECT_SUMMARY function. Using metabolomic approaches, we show that downregulation of taurine PR:PROJECT_SUMMARY metabolism is crucial for NLRP3 inflammasome activation. Following NLRP3 PR:PROJECT_SUMMARY activation stimuli, taurine rapidly egresses to the extracellular compartment. PR:PROJECT_SUMMARY Taurine efflux is facilitated primarily by the volume-regulated anion channel PR:PROJECT_SUMMARY (VRAC). Loss of intracellular taurine impairs sodium-potassium ATPase pump PR:PROJECT_SUMMARY activity, promoting ionic dysregulation and disrupting ionic fluxes. Inhibiting PR:PROJECT_SUMMARY VRAC, or supplementation of taurine, restores the ionic balance, abrogates PR:PROJECT_SUMMARY IL-1beta release and reduces cellular cytotoxicity in macrophages. We further PR:PROJECT_SUMMARY demonstrate that the protective effect of taurine is diminished when PR:PROJECT_SUMMARY sodium-potassium ATPase is inhibited, highlighting the pump’s role in PR:PROJECT_SUMMARY taurine-mediated protection. Finally, taurine metabolism is significantly PR:PROJECT_SUMMARY associated with the development of tuberculosis-associated immune reconstitution PR:PROJECT_SUMMARY inflammatory syndrome, a systemic hyperinflammatory condition known to be PR:PROJECT_SUMMARY mediated by inflammasome activation. Altogether, we identified a critical PR:PROJECT_SUMMARY metabolic pathway that modulates inflammasome activation and drives disease PR:PROJECT_SUMMARY pathogenesis. PR:INSTITUTE Imperial College London PR:DEPARTMENT Department of Infectious Disease PR:LABORATORY Lai's Lab PR:LAST_NAME Rossi-Smith PR:FIRST_NAME Peter PR:ADDRESS Hammersmith Campus, London, London, W12 0NN, United Kingdom PR:EMAIL p.rossi@imperial.ac.uk PR:PHONE 07860694004 PR:FUNDING_SOURCE This work was supported by an MRC CDA fellowship (MR/R008922/1) to R.P.J.L. and PR:FUNDING_SOURCE in part by the NIHR Imperial Biomedical Research Centre and an NIH R01 grant PR:FUNDING_SOURCE (5R01AI145436) to R.J.W. and R.P.J.L. D.C.T. is supported by a Wellcome-Beit PR:FUNDING_SOURCE Prize Trust Clinical Research Career Development Fellowship and the Burman Fund PR:FUNDING_SOURCE from Imperial College London. J.P.G. is supported by MRC research grant PR:FUNDING_SOURCE (MR/W028867/1). A.E.D. is supported by an MRC CDA fellowship (MR/V009591/1). PR:FUNDING_SOURCE R.J.W., M.S.S. and J.I.M. are supported by The Francis Crick Institute, which PR:FUNDING_SOURCE receives its core funding from Cancer Research UK (CC2206), the UK Medical PR:FUNDING_SOURCE Research Council (CC2206), and the Wellcome Trust (CC2206). T.E. and C.W. PR:FUNDING_SOURCE acknowledge funding from the BBSRC grant (BB/W002345/1). T.E. acknowledges PR:FUNDING_SOURCE partial support from UKRI BBSRC grant BB/T007974/1, European Union projects PR:FUNDING_SOURCE HUMAN (EC101073062) and BiACEM (EC101079370). G.M. was supported by the Wellcome PR:FUNDING_SOURCE Trust (098316, 214321/Z/18/Z, and 203135/Z/16/Z) and the South African Research PR:FUNDING_SOURCE Chairs Initiative of the Department of Science and Technology and National PR:FUNDING_SOURCE Research Foundation (NRF) of South Africa (Grant no. 64787). The funders had no PR:FUNDING_SOURCE role in the study design, data collection, data analysis, data interpretation, PR:FUNDING_SOURCE or writing of this report. The opinions, findings and conclusions expressed in PR:FUNDING_SOURCE this manuscript reflect those of the authors alone. This research was funded, in PR:FUNDING_SOURCE part, by the Wellcome Trust. For the purpose of open access, the authors have PR:FUNDING_SOURCE applied a CC BY public copyright license to any Author Accepted Manuscript PR:FUNDING_SOURCE version arising from this submission. PR:CONTRIBUTORS Dr. Rachel Lai #STUDY ST:STUDY_TITLE Metabolic changes in NLRP3 knockout and VRAC knockdown THP-1 derived macrophages ST:STUDY_TITLE following NLRP3 inflammasome activation ST:STUDY_TYPE Exploratory MS ST:STUDY_SUMMARY Using metabolomic approaches, we show that downregulation of taurine metabolism ST:STUDY_SUMMARY is crucial for NLRP3 inflammasome activation. Following NLRP3 activation ST:STUDY_SUMMARY stimuli, taurine rapidly egresses to the extracellular compartment. Taurine ST:STUDY_SUMMARY efflux is facilitated primarily by the volume-regulated anion channel (VRAC). ST:STUDY_SUMMARY Inhibiting VRAC, or supplementation of taurine, restores the ionic balance, ST:STUDY_SUMMARY abrogates IL-1beta release and reduces cellular cytotoxicity in THP-1 derived ST:STUDY_SUMMARY macrophages. ST:INSTITUTE Imperial College London ST:LAST_NAME Rossi-Smith ST:FIRST_NAME Peter ST:ADDRESS Hammersmith Campus, London, London, W12 0NN, United Kingdom ST:EMAIL p.rossi@imperial.ac.uk ST:PHONE 07860694004 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN THP-1 cell line #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 - THP-1_1 Treatment:Control | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_1.d SUBJECT_SAMPLE_FACTORS - THP-1_2 Treatment:Control | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_2.d SUBJECT_SAMPLE_FACTORS - THP-1_3 Treatment:Control | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_3.d SUBJECT_SAMPLE_FACTORS - THP-1_4 Treatment:Control | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_4.d SUBJECT_SAMPLE_FACTORS - THP-1_5 Treatment:Control | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_5.d SUBJECT_SAMPLE_FACTORS - THP-1_6 Treatment:LPS+ATP | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_6.d SUBJECT_SAMPLE_FACTORS - THP-1_7 Treatment:LPS+ATP | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_7.d SUBJECT_SAMPLE_FACTORS - THP-1_8 Treatment:LPS+ATP | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_8.d SUBJECT_SAMPLE_FACTORS - THP-1_9 Treatment:LPS+ATP | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_9.d SUBJECT_SAMPLE_FACTORS - THP-1_10 Treatment:LPS+ATP | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_10.d SUBJECT_SAMPLE_FACTORS - THP-1_11 Treatment:LPS+NG | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_11.d SUBJECT_SAMPLE_FACTORS - THP-1_12 Treatment:LPS+NG | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_12.d SUBJECT_SAMPLE_FACTORS - THP-1_13 Treatment:LPS+NG | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_13.d SUBJECT_SAMPLE_FACTORS - THP-1_14 Treatment:LPS+NG | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_14.d SUBJECT_SAMPLE_FACTORS - THP-1_15 Treatment:LPS+NG | Sample source:WT RAW_FILE_NAME(RAW file name)=THP-1_15.d SUBJECT_SAMPLE_FACTORS - THP-1_31 Treatment:Control | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_31.d SUBJECT_SAMPLE_FACTORS - THP-1_32 Treatment:Control | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_32.d SUBJECT_SAMPLE_FACTORS - THP-1_33 Treatment:Control | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_33.d SUBJECT_SAMPLE_FACTORS - THP-1_34 Treatment:Control | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_34.d SUBJECT_SAMPLE_FACTORS - THP-1_35 Treatment:Control | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_35.d SUBJECT_SAMPLE_FACTORS - THP-1_36 Treatment:LPS+ATP | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_36.d SUBJECT_SAMPLE_FACTORS - THP-1_37 Treatment:LPS+ATP | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_37.d SUBJECT_SAMPLE_FACTORS - THP-1_38 Treatment:LPS+ATP | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_38.d SUBJECT_SAMPLE_FACTORS - THP-1_39 Treatment:LPS+ATP | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_39.d SUBJECT_SAMPLE_FACTORS - THP-1_40 Treatment:LPS+ATP | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_40.d SUBJECT_SAMPLE_FACTORS - THP-1_41 Treatment:LPS+NG | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_41.d SUBJECT_SAMPLE_FACTORS - THP-1_42 Treatment:LPS+NG | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_42.d SUBJECT_SAMPLE_FACTORS - THP-1_43 Treatment:LPS+NG | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_43.d SUBJECT_SAMPLE_FACTORS - THP-1_44 Treatment:LPS+NG | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_44.d SUBJECT_SAMPLE_FACTORS - THP-1_45 Treatment:LPS+NG | Sample source:NLRP3 KO RAW_FILE_NAME(RAW file name)=THP-1_45.d SUBJECT_SAMPLE_FACTORS - THP-1_46 Treatment:Control | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_46.d SUBJECT_SAMPLE_FACTORS - THP-1_47 Treatment:Control | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_47.d SUBJECT_SAMPLE_FACTORS - THP-1_48 Treatment:Control | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_48.d SUBJECT_SAMPLE_FACTORS - THP-1_49 Treatment:Control | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_49.d SUBJECT_SAMPLE_FACTORS - THP-1_50 Treatment:Control | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_50.d SUBJECT_SAMPLE_FACTORS - THP-1_51 Treatment:LPS+ATP | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_51.d SUBJECT_SAMPLE_FACTORS - THP-1_52 Treatment:LPS+ATP | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_52.d SUBJECT_SAMPLE_FACTORS - THP-1_53 Treatment:LPS+ATP | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_53.d SUBJECT_SAMPLE_FACTORS - THP-1_54 Treatment:LPS+ATP | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_54.d SUBJECT_SAMPLE_FACTORS - THP-1_55 Treatment:LPS+ATP | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_55.d SUBJECT_SAMPLE_FACTORS - THP-1_56 Treatment:LPS+NG | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_56.d SUBJECT_SAMPLE_FACTORS - THP-1_57 Treatment:LPS+NG | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_57.d SUBJECT_SAMPLE_FACTORS - THP-1_58 Treatment:LPS+NG | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_58.d SUBJECT_SAMPLE_FACTORS - THP-1_59 Treatment:LPS+NG | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_59.d SUBJECT_SAMPLE_FACTORS - THP-1_60 Treatment:LPS+NG | Sample source:VRAC KD RAW_FILE_NAME(RAW file name)=THP-1_60.d #COLLECTION CO:COLLECTION_SUMMARY Following in vitro experiments, mBMDM metabolites were quenched by washing the CO:COLLECTION_SUMMARY cells twice with ice-cold AUTOMacs Rinsing Solution (Miltenyi Biotec), before a CO:COLLECTION_SUMMARY methanol (10767665, Fisher Chemical):water (10505904, Fisher Chemical) (4:1 v/v) CO:COLLECTION_SUMMARY solution was added and macrophages were gently scrapped. Lysed macrophages were CO:COLLECTION_SUMMARY re-suspended in chloroform (10615492, Fisher Chemical) and submitted to 3 CO:COLLECTION_SUMMARY cycles: vortex for 0.5 min and placed on ice for 5 min. Following the last CO:COLLECTION_SUMMARY vortexing cycle the samples were stored at -80°C for no less than 12 hours. CO:COLLECTION_PROTOCOL_FILENAME LC-MS_protocol.pdf CO:SAMPLE_TYPE Macrophages CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY All inflammasome activation reagents were sourced from InvivoGen, unless TR:TREATMENT_SUMMARY otherwise specified. NLRP3 inflammasome activation was induced in THP-1 derived TR:TREATMENT_SUMMARY macrophages by priming with 500 ng/mL LPS (tlrl-peklps) for 3.5 hours, followed TR:TREATMENT_SUMMARY by stimulation with either 5 mM ATP (tlrl-atpl) or 20 µM nigericin (N7143, TR:TREATMENT_SUMMARY Sigma-Aldrich) for ~45 minutes #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After overnight incubation in -80°C, water was added to generate a biphasic SP:SAMPLEPREP_SUMMARY solution with a final dilution of 3:2:4 (v/v) chloroform:water:methanol. The SP:SAMPLEPREP_SUMMARY samples were then vortexed and centrifuged at 14,000 rpm for 10 min. at 0°C. SP:SAMPLEPREP_SUMMARY The top layer containing polar metabolites (avoiding the interface) was SP:SAMPLEPREP_SUMMARY concentrated using a SpeedVac. The dried samples were resuspended in 75 µL of SP:SAMPLEPREP_SUMMARY 30% methanol and 2% acetonitrile (10001334, Fisher Chemical) and stored at SP:SAMPLEPREP_SUMMARY -80°C until metabolomics analyses were carried out. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed using an Agilent 1290 Infinity II UHPLC coupled with CH:CHROMATOGRAPHY_SUMMARY Agilent 6546 LC/QTOF. The system was equipped with an Agilent Poroshell 120 CH:CHROMATOGRAPHY_SUMMARY HILIC-Z column (2.1 x 150 mm, 2.1 µm). A 2 µL sample volume was injected, and CH:CHROMATOGRAPHY_SUMMARY the chromatographic separation was performed at 15°C with a flow rate of 400 CH:CHROMATOGRAPHY_SUMMARY µL/min using an elution gradient. Mobile phases A (20 mM ammonium acetate, 5 CH:CHROMATOGRAPHY_SUMMARY µM medronic acid, pH 9.3) and B (acetonitrile) were used with the following CH:CHROMATOGRAPHY_SUMMARY gradient: 0-1 min, 85% B; 1-8 min, 75% B; 8-12 min, 60% B; 12-19.10 min, 10% B; CH:CHROMATOGRAPHY_SUMMARY 19.10-24 min, 85% B. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C8 (150 x 2.1 mm, 2.7 µm) CH:SOLVENT_A 100% Water; 20 mM Ammonium acetate; 5 µM Medronic acid (pH 9.3) CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT 0-1 min, 85% B; 1-8 min, 75% B; 8-12 min, 60% B; 12-19.10 min, 10% B; 19.10-24 CH:FLOW_GRADIENT min, 85% B CH:FLOW_RATE 400 µL/min CH:COLUMN_TEMPERATURE 15°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6546 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Parameters of the LC/QTOF were the following: gas temperature, 225°C; drying gas MS:MS_COMMENTS flow, 9 L/min; nebulizer gas pressure, 30 psi; sheath gas temperature, 375°C; MS:MS_COMMENTS capillary voltage, 3000 V; nozzle voltage, 500 V, fragmentor 100 V, skimmer 45 MS:MS_COMMENTS V, and octupole 1 radio frequency volts peak to peak (Vpp) 750 V. The data was MS:MS_COMMENTS acquired in low mass range (1700 m/z). Analytical standards from an in-house MS:MS_COMMENTS library were injected into the LC-MS system to verify retention times and MS/MS MS:MS_COMMENTS fragmentation patterns. MS:MS_RESULTS_FILE ST004113_AN006820_Results.txt UNITS:Area Has m/z:Yes Has RT:Yes RT units:Minutes #END