#METABOLOMICS WORKBENCH Peter_RS_20250813_010232 DATATRACK_ID:6275 STUDY_ID:ST004115 ANALYSIS_ID:AN006822 PROJECT_ID:PR002586 VERSION 1 CREATED_ON August 14, 2025, 2:03 am #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 murine macrophages following NLRP3 inflammasome activation ST:STUDY_TYPE Exploratory MS ST:STUDY_SUMMARY The intersection of immunology and metabolism, known as immunometabolism, ST:STUDY_SUMMARY explores the interactions between immune responses and metabolic changes. ST:STUDY_SUMMARY Inflammasomes form an integral part of the innate immune system and are equipped ST:STUDY_SUMMARY with NLR or ALR receptors capable of detecting a wide array of stimuli triggered ST:STUDY_SUMMARY by infections or cellular damage. Upon activation, these inflammasomes are ST:STUDY_SUMMARY involved in the release of inflammatory cytokines and can trigger a regulated ST:STUDY_SUMMARY type of cell death known as pyroptosis. Like other immune responses, ST:STUDY_SUMMARY inflammasome activation also induces changes in metabolic pathways such as the ST:STUDY_SUMMARY tricarboxylic acid (TCA) cycle. However, the role of other metabolic pathways in ST:STUDY_SUMMARY response to activation of inflammasomes remains less explored. Here, by ST:STUDY_SUMMARY employing a metabolomic approach on murine macrophages, we found that activation ST:STUDY_SUMMARY of inflammasomes (NLRP3, AIM2 or NLRC4) induced metabolic shifts not only within ST:STUDY_SUMMARY the TCA cycle, but also extends its impact to Sulphur metabolism. Furthermore, ST:STUDY_SUMMARY through a rigorous cross-species analysis that compared human and mouse ST:STUDY_SUMMARY responses (shown in Taurine transport is a critical modulator of ionic fluxes ST:STUDY_SUMMARY during NLRP3 inflammasome activation project), we uncovered a notable ST:STUDY_SUMMARY downregulation of taurine metabolism following NLRP3 activation. This intriguing ST:STUDY_SUMMARY discovery highlighted a conserved regulatory mechanism and identified ST:STUDY_SUMMARY intracellular depletion of taurine and hypotaurine as a putative checkpoint in ST:STUDY_SUMMARY NLRP3 activation pathway. 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 Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 #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 - WT2_1 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_1.d SUBJECT_SAMPLE_FACTORS - WT2_2 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_2.d SUBJECT_SAMPLE_FACTORS - WT2_3 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_3.d SUBJECT_SAMPLE_FACTORS - WT2_4 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_4.d SUBJECT_SAMPLE_FACTORS - WT2_5 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_5.d SUBJECT_SAMPLE_FACTORS - WT2_6 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_6.d SUBJECT_SAMPLE_FACTORS - WT2_7 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_7.d SUBJECT_SAMPLE_FACTORS - WT2_8 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_8.d SUBJECT_SAMPLE_FACTORS - WT2_9 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_9.d SUBJECT_SAMPLE_FACTORS - WT2_10 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_10.d SUBJECT_SAMPLE_FACTORS - WT2_11 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_11.d SUBJECT_SAMPLE_FACTORS - WT2_12 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_12.d SUBJECT_SAMPLE_FACTORS - WT2_13 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_13.d SUBJECT_SAMPLE_FACTORS - WT2_14 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_14.d SUBJECT_SAMPLE_FACTORS - WT2_15 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_15.d SUBJECT_SAMPLE_FACTORS - WT2_16 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_16.d SUBJECT_SAMPLE_FACTORS - WT2_17 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_17.d SUBJECT_SAMPLE_FACTORS - WT2_18 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_18.d SUBJECT_SAMPLE_FACTORS - WT2_19 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_19.d SUBJECT_SAMPLE_FACTORS - WT2_20 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_20.d SUBJECT_SAMPLE_FACTORS - WT2_21 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_21.d SUBJECT_SAMPLE_FACTORS - WT2_22 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_22.d SUBJECT_SAMPLE_FACTORS - WT2_23 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_23.d SUBJECT_SAMPLE_FACTORS - WT2_24 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_24.d SUBJECT_SAMPLE_FACTORS - WT2_25 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_25.d SUBJECT_SAMPLE_FACTORS - WT2_26 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_26.d SUBJECT_SAMPLE_FACTORS - WT2_27 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_27.d SUBJECT_SAMPLE_FACTORS - WT2_28 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_28.d SUBJECT_SAMPLE_FACTORS - WT2_29 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_29.d SUBJECT_SAMPLE_FACTORS - WT2_30 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=WT2_30.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 mBMDM by TR:TREATMENT_SUMMARY priming with 500 ng/mL LPS (tlrl-peklps) for 3.5 hours, followed by stimulation TR:TREATMENT_SUMMARY with either 5 mM ATP (tlrl-atpl) or 20 uM nigericin (N7143, Sigma-Aldrich) for TR:TREATMENT_SUMMARY ~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 POSITIVE MS:MS_COMMENTS Parameters of the LC/QTOF were the following: gas temperature, 225°C; drying MS:MS_COMMENTS gas flow, 9 L/min; nebulizer gas pressure, 30 psi; sheath gas temperature, MS:MS_COMMENTS 375°C; capillary voltage, 3000 V; nozzle voltage, 500 V, fragmentor 100 V, MS:MS_COMMENTS skimmer 45 V, and octupole 1 radio frequency volts peak to peak (Vpp) 750 V. The MS:MS_COMMENTS data was acquired in low mass range (1700 m/z). MS:MS_RESULTS_FILE ST004115_AN006822_Results.txt UNITS:Area Has m/z:Yes Has RT:Yes RT units:Minutes #END