MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12917/m_MTBLS12917_LC-MS_positive_hilic_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12917/m_MTBLS12917_LC-MS_negative_hilic_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12917/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12917/s_MTBLS12917.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12917/a_MTBLS12917_LC-MS_negative_hilic_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12917/a_MTBLS12917_LC-MS_positive_hilic_metabolite_profiling.txtprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12917<p>Peaks were annotated by comparison with authentic standards. Relative metabolite concentrations were quantified by manually integrating peak areas for each metabolite. If major chromatographic shifts occurred between runs, peak alignment was performed using the OBI-Warp algorithm with a PBQC sample as a reference65. Peak areas were exported as a csv matrix and data was pretreated using MetaboAnalyst66–68 to account for biological, experimental, and instrument variations. Pretreatment included a natural log transformation, Pareto scaling, and normalising to the median metabolite abundance within each sample69. Statistical comparisons were then made using unpaired Student’s t-test (α = 0.05), with false discovery rate (FDR) correction using the Benjamini-Hochberg method70. Enrichment analysis was performed using the KEGG metabolite library in MetaboAnalyst.</p>MetaboLightsPublicLiquid Chromatography MS - positive - HILICLiquid Chromatography MS - negative - HILIC<p>Dried metabolite extracts were resuspended in 200 µL of 80% acetonitrile prior to liquid chromatography (LC)-MS. Samples were analsed using a Thermo Scientific Vanquish Quaternary Pumps LC System™ coupled to a Thermo Scientific Orbitrap IQ-X Tribrid Mass Spectrometer™. Five μL of each sample were injected onto a SeQuant® ZIC®-pHILIC column (5 µm pore size, 150 x 4.6 mm, PEEK-coated; Merck Millipore) preceded by a SeQuant® ZIC®-pHILIC guard column (20 x 2.1 mm, PEEK-coated; Merck Millipore) using a Thermo Scientific™ Vanquish™ Ultra High-Performance Liquid Chromatography (UHPLC) system. Mobile phases were 20 mM ammonium carbonate pH 9 (Eluent A), and acetonitrile (Eluent B) with a flow rate of 0.3 ml/min. The gradient elution profile was as follows: 0 min 80% B; 15.5 min 50% B; 17.5 min 30% B; 18.5 min 5% B; 21 min maintained at 5% B; 23 min 80% B; 30 maintained at 80% B.Please update this protocol description</p>Vitronectin metabolically programs pro-fibrotic macrophages in 3D cultures and idiopathic pulmonary fibrosis.Sean CutterKatrina BingerLa Trobe UniversityMacrophagesmass spectrometry assay<p>Washed cell pellets were then washed once with ice-cold ddH2O, and metabolites were extracted from the resulting cell pellets by adding 500 µL of methyl-tert-butyl ether (MTBE) and methanol (3:1, v/v). Samples were sonicated for 10 min to facilitate extraction. Biphasic partitioning was initiated by adding 250 µL of a methanol:water mixture (1:3, v/v), followed by shaking on an Eppendorf Thermomixer C for 30 min at 4 °C and 2000 RPM. This resulted in a final solvent composition of 6:2:3 MTBE:methanol:water (750 μL). Samples were centrifuged at 16,000 x g for 15 min, 4 °C to separate phases. The apolar phase (top layer) was removed, and the polar (bottom layer) containing water-soluble metabolites was retained, centrifuged again, and dried under a nitrogen manifold.Please update this protocol description</p>Mus musculushttps://www.ebi.ac.uk/metabolights/MTBLS12917Katrina Binger. La Trobe University. La Trobe University, Dept Biochemistry and Chemistry, Bundoora VIC 3086, Australia. k.binger@latrobe.edu.au.Mark Hulett. La Trobe University.Gang Liu. University of Technology Sydney.Eleanor Saunders. The University of Melbourne.Phillip Hansbro. University of Technology Sydney.Vinzenz Hofferek. The University of Melbourne.Mark Wright. Monash University.Thomas Soerianto. The University of Melbourne.Erin McGowan. The University of Melbourne.Nicholas Reynolds. RMIT University.Ivan Poon. La Trobe University.Tien Nguyen. La Trobe University.Kaitlyn Ritchie. La Trobe University.Malcolm McConville. The University of Melbourne.Sean Cutter. La Trobe University. La Trobe University, Department Biochemistry and Chemistry, Bundoora VIC 3086, Australia. s.cutter@latrobe.edu.au.<p>Raw LC-MS data was processed using El-MAVEN (Agrawal et al Meth Mol Biol 1978) for peak integration and quantification as follows.</p>TreatmentCulture dimensionk.binger@latrobe.edu.auS.Cutter@latrobe.edu.au<p>BMDMs were cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% FBS, 2 mM L-Glutamine (Sigma-Aldrich), 1% penicillin/streptomycin (Sigma-Aldrich) and 50 µM 2-mercaptoethanol (Sigma-Aldrich) at 37°C, 5% CO2. For 3D culture, BMDMs cells were suspended in a 1:1 mixture with collagen (5 mg/mL; 5074, PureCol® Bovine Type I Collagen, Advanced Biomatrix). Samples were incubated for 30 min in 96-wells or 60 min in 24-wells at 37°C, 5% CO2 to ensure complete gelation of the collagen matrix. A total cell:collagen volume of 150 μL was used for 96-well plate assays, and 500 μL for 24-well plates, with an additional 100 μL and 500 μL of culture media added respectively after the gelation time to ensure hydration of gels and the provision of sufficient nutrients. This resulted in a final collagen concentration of 2.5 mg/mL, cell density of 1 x 106 cells/mL, in total volumes of 250 μL (96-well) and 1000 μL (24-well). ECM proteins were added to the cell-collagen mixture prior to gelation, at the following final concentrations: human vitronectin (5 μg/mL; 10424-H08H, Sino Biological). Control conditions included BMDMs seeded directly onto standard 2D tissue culture-treated plastic plates, or onto plates pre-coated with a monolayer of the respective ECM protein(s). For these 2D-coated controls, wells were incubated with 0.5 mg/mL collagen (5074, PureCol® Bovine Type I Collagen, Advanced Biomatrix) diluted in 0.02 M acetic acid for 2 hrs at 37°C, followed by washing twice with phosphate buffered saline (PBS) prior to cell seeding. If required, other ECM proteins were included in this coating solution at the same concentrations as described above for 3D cultures. For 2D controls (plastic plates and ECM-coated), cells were seeded at the same density and total media volume as for 3D conditions. Macrophage activation was induced 3 hours after seeding/gelation of the 3D cultures by the supplementation of culture media with either 10 ng/mL LPS (tlrl-3pelps, Invivogen) and 10 ng/mL murine IFNγ (ab9922, Abcam), or 10 ng/mL murine IL-4 (214-14, PeproTech) and 10 ng/mL IL-13 (210-13, PeproTech). Unactivated BMDMs were maintained in standard media. BMDMs were isolated from 3D scaffolds by enzymatic digestion with 1.6 mg/mL collagenase (Type IV; 17104019, ThermoFisher) for 25 min at 37°C, with gentle agitation every 5 min, with gentle pipetting after the incubation period to disrupt any remaining gel fragments. For consistency, 2D control samples were treated with the same concentration of collagenase under identical conditions. Cells adhered to 2D plates were detached by the addition of PBS supplemented with 0.5% v/v FBS and 2 mM EDTA. Single cell suspensions were then centrifuged at 300 x g for 5 min and cell metabolism was quenched by resuspension of cell pellets in ice-cold PBS. </p>MetabolomicsMus musculusPhenotypeuntargeted analysisThermo Scientific Vanquish Flex UHPLC SystemThermo Scientific Orbitrap IQ-X Tribriduntargeted metabolitesexperimental blankmacrophage activationMacrophagesMacrophagePhenotypeMus musculusuntargeted analysisThermo Scientific Vanquish Flex UHPLC SystemThermo Scientific Orbitrap IQ-X Tribriduntargeted metabolitesexperimental blankmacrophage activationMacrophagesMacrophage<p>Mass spectrometry data were acquired using a Thermo Scientific Orbitrap IQ-X Tribrid Mass Spectrometer in both positive and negative mode using H-ESI with the following parameters: spray voltage was set 3500 V (positive) or 3200 V (negative); sheath gas at 50 Arbitrary (Arb) units; auxiliary gas at 10 Arb units; and sweep gas at 1 Arb unit. The ion transfer tube temperature was set to 300°C, and the vaporizer temperature to 400°C. Full MS scans were acquired using the Orbitrap detector at a resolution of 120,000 FWHM, with RF lens set to 60%. Prior to data collection, 10 blank injections were performed followed by 5 quality control samples. Pooled biological quality control samples (PBQC) were interspersed between every 5 samples to monitor instrument performance and correct for run-to-run variability. </p>GTPinosineglutathione disulfideL-tryptophanL-threonic acidADPUTPGDPhypoxanthinecis-aconitic acidL-asparagineUDPL-glutaminecytidinetaurine5-oxo-L-proline(R)-lactic acidL-pipecolic acidN-acetyl-L-aspartic acidUDP-N-acetyl-?-D-glucosamineIMPcoenzyme Aadenosine(S)-malic acidsedoheptulose 7-phosphatecreatineL-aspartic acidsuccinic acidO-acetyl-L-serineO-phosphoethanolamineL-alanineL-isoleucineGlycero-3-phosphocholineL-phenylalanineUDP-?-D-glucoseglutathioneL-leucineL-methioninetetradecanoic acid2'-deoxyuridineitaconic aciduridineNADHpyridoxine2-oxoglutaric acidL-prolinecyclic ADP-?-D-ribosefumaric acidL-glutamic acidL-threonineNADP+cytidine 5'-monophosphateGDP-?-D-mannoseL-valineadenineL-tyrosineuracilacetyl-CoAisocitric acidATPCTPNAD+N-phosphocreatinefalseComparative non-targeted LC-MS metabolomic profiling of murine bone marrow-derived macrophages cultured in 2D versus 3D<p>Macrophages are key drivers of inflammatory and fibrotic diseases and their activation is shaped by interactions in their tissue microenvironment. However, dissecting the processes that drive immunopathology has proved challenging as traditional 2D culture methods fail to capture the complex molecular environment that macrophages inhabit in vivo. To address this, we generated a 3D in vitro model to better mimic the in vivo biophysical microenvironment. We show that the extracellular matrix (ECM) protein vitronectin promotes a novel pro-fibrotic macrophage phenotype in 3D that is characterised by increased expression of the NAD+ ectoenzyme CD38, elevated glycolysis and mitochondrial metabolism, and synthesis of the immunomodulatory metabolite itaconate. This is validated in vivo where vitronectin-deficient mice were protected from an experimental model of idiopathic pulmonary fibrosis. Thus, we uncover a novel link between the composition of tissue niches and macrophage pro-fibrotic function via altered metabolic reprogramming. </p>2026-06-082025-09-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:17191CHEBI:16857CHEBI:15846CHEBI:30797CHEBI:16908CHEBI:31445CHEBI:17053CHEBI:17295CHEBI:16919CHEBI:17196CHEBI:17287CHEBI:16704CHEBI:17368CHEBI:16643CHEBI:16708CHEBI:17202CHEBI:32805CHEBI:46229CHEBI:30913CHEBI:17361CHEBI:17568CHEBI:15721CHEBI:15908CHEBI:42111CHEBI:15741CHEBI:30915CHEBI:16414CHEBI:16264CHEBI:30838CHEBI:16709CHEBI:15820CHEBI:16450CHEBI:30887CHEBI:18183CHEBI:16335CHEBI:17203CHEBI:21547CHEBI:17895CHEBI:17677CHEBI:15713CHEBI:17552CHEBI:16828CHEBI:16977CHEBI:15346CHEBI:15603CHEBI:16856CHEBI:17553CHEBI:194386CHEBI:28875CHEBI:17596CHEBI:17562CHEBI:18012CHEBI:17981CHEBI:16015CHEBI:17659CHEBI:18009CHEBI:15996CHEBI:15891CHEBI:16761CHEBI:17858CHEBI:18050CHEBI:15422CHEBI:15351