#METABOLOMICS WORKBENCH Peter_RS_20250811_073226 DATATRACK_ID:6271 STUDY_ID:ST004111 ANALYSIS_ID:AN006821 PROJECT_ID:PR002586 VERSION 1 CREATED_ON August 14, 2025, 3:39 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 human macrophages following NLRP3 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 human macrophages, we found that activation ST:STUDY_SUMMARY of inflammasomes NLRP3 induced metabolic shifts not only within the TCA cycle, ST:STUDY_SUMMARY but also extends its impact to Sulphur metabolism, more specifically ST:STUDY_SUMMARY downregulation of the taurine metabolism. 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 Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - 220119_PR211125_h01 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h01.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h02 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h02.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h03 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h03.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h04 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h04.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h05 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h05.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h06 Treatment:ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h06.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h07 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h07.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h08 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h08.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h09 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h09.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h10 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h10.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h11 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h11.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h12 Treatment:NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h12.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h13 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h13.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h14 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h14.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h15 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h15.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h16 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h16.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h17 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h17.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h18 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h18.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h19 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h19.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h20 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h20.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h21 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h21.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h22 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h22.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h23 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h23.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h24 Treatment:LPS+ATP | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h24.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h25 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h25.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h26 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h26.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h27 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h27.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h28 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h28.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h29 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h29.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h30 Treatment:LPS+NG | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h30.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h31 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h31.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h32 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h32.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h33 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h33.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h34 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h34.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h35 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h35.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_h36 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_h36.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_PBQC_h5POS Treatment:MSMS_PBQC_Pos | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_PBQC_h5POS.raw SUBJECT_SAMPLE_FACTORS - 220119_PR211125_PBQC_h6NEG Treatment:MSMS_PBQC_Neg | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=220119_PR211125_PBQC_h6NEG.raw #COLLECTION CO:COLLECTION_SUMMARY Following in vitro experiments, hBMM 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 #TREATMENT TR:TREATMENT_SUMMARY All inflammasome activation reagents were sourced from InvivoGen, unless TR:TREATMENT_SUMMARY otherwise specified. NLRP3 inflammasome activation was induced in hBMM 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 uL 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. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed using a Dionex UltiMate Liquid Chromatography (LC) system CH:CHROMATOGRAPHY_SUMMARY (Thermo Scientific) with a ZIC-pHILIC (150 mm x 4.6 mm, 5 µm particle) CH:CHROMATOGRAPHY_SUMMARY column (Merck Sequant). The mobile phase contained 20 mM ammonium carbonate in CH:CHROMATOGRAPHY_SUMMARY water – solvent A (Optima HPLC grade, Sigma Aldrich), and acetonitrile – CH:CHROMATOGRAPHY_SUMMARY solvent B (Optima HPLC grade, Sigma Aldrich). Samples were separated using an CH:CHROMATOGRAPHY_SUMMARY elution gradient: 15 min of 4:1 (Solvent A:Solvent B), 5 min 95:5 (Solvent CH:CHROMATOGRAPHY_SUMMARY A:Solvent B) and 5 min re-equilibration. The samples were kept at 4°C during CH:CHROMATOGRAPHY_SUMMARY the whole run, the mobile phase had a flow rate of 300 uL/min and the column CH:CHROMATOGRAPHY_SUMMARY temperature was 25°C CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Merck SeQuant ZIC-pHILIC (150 x 4.6 mm, 5 µm) CH:SOLVENT_A 100% Water; 20 mM Ammonium carbonate CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT 15 min of 4:1 (Solvent A:Solvent B), 5 min 95:5 (Solvent A:Solvent B) and 5 min CH:FLOW_GRADIENT re-equilibration. CH:FLOW_RATE 300 µL/min CH:COLUMN_TEMPERATURE 25°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS The samples were analyzed using a Q Exactive Orbitrap (Thermo Scientific) mass MS:MS_COMMENTS spectrometer (MS) with a Heated electrospray ionization probe using both MS:MS_COMMENTS positive/negative polarities. The spray voltage was 3.5 and 3.2 kV for positive MS:MS_COMMENTS and negative modes, respectively; 320°C for the probe; 30 and 5 arbitrary units MS:MS_COMMENTS for the sheath and auxiliary gases, respectively; 70 – 1050 m/z full scan MS:MS_COMMENTS range with AGC target and balanced and high resolution (3 x 106 and 70,000). The MS:MS_COMMENTS instrument was calibrated using Calmix solution (Thermo Scientific) in both ESI MS:MS_COMMENTS polarities. Lock-mass correction was applied to each run, using ubiquitous MS:MS_COMMENTS low-mass contaminants. MS:MS_RESULTS_FILE ST004111_AN006821_Results.txt UNITS:Area Has m/z:Yes Has RT:Yes RT units:Minutes #END