#METABOLOMICS WORKBENCH Peter_RS_20250813_021052 DATATRACK_ID:6276 STUDY_ID:ST004114 ANALYSIS_ID:AN006819 PROJECT_ID:PR002586 VERSION 1 CREATED_ON August 14, 2025, 11:45 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 AIM2 and NLRC4 inflammasomes ST:STUDY_TITLE 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 - Mu_Other_Infla_1 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_1.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_2 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_2.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_3 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_3.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_4 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_4.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_5 Treatment:Control | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_5.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_6 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_6.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_7 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_7.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_8 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_8.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_9 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_9.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_10 Treatment:LPS | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_10.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_11 Treatment:AIM2 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_11.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_12 Treatment:AIM2 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_12.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_13 Treatment:AIM2 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_13.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_14 Treatment:AIM2 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_14.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_15 Treatment:AIM2 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_15.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_16 Treatment:NLRC4 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_16.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_17 Treatment:NLRC4 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_17.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_18 Treatment:NLRC4 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_18.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_19 Treatment:NLRC4 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_19.d SUBJECT_SAMPLE_FACTORS - Mu_Other_Infla_20 Treatment:NLRC4 | Sample source:Macrophages RAW_FILE_NAME(RAW file name)=Mu_Other_Infla_20.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. To activate the AIM2 inflammasome, mBMDM were primed with TR:TREATMENT_SUMMARY 500 ng/mL LPS for 2 hours, then transfected, using LyoVecTM (lyec-1), with a DNA TR:TREATMENT_SUMMARY complex containing 5 µg/mL poly(dA:dT) (tlrl-patn-1) for 4 hours (14). NLRC4 TR:TREATMENT_SUMMARY inflammasome was activated by priming macrophages with LPS for 2 hours before TR:TREATMENT_SUMMARY transfecting 1 µg/mL of Pseudomonas aeruginosa flagellin (tlrl-pafla), using TR:TREATMENT_SUMMARY LyoVecTM as the transfecting agent. Following transfection, cells were incubated TR:TREATMENT_SUMMARY for 4 hours. #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 CH:CHROMATOGRAPHY_SUMMARY 400uL/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 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). MS:MS_RESULTS_FILE ST004114_AN006819_Results.txt UNITS:Area Has m/z:Yes Has RT:Yes RT units:Minutes #END