Summary of Study ST000657

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000462. The data can be accessed directly via it's Project DOI: 10.21228/M8BG7G This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST000657
Study TitlemTOR regulates metabolic adaptation of APCs in the lung microenvironment and controls the outcome of allergic inflammation
Study SummaryAntigen presenting cells (APCs) occupy diverse anatomical tissues, but their tissue-restricted homeostasis remains poorly understood. Here, working in mouse models of inflammation, we found that mTOR-dependent metabolic adaptation was required at discrete locations. mTOR was dispensable for DC homeostasis in secondary lymphoid tissues, but necessary to regulate cellular metabolism and accumulation of CD103+ DCs and alveolar macrophages in lung. Moreover, whilst numbers of mTOR-deficient lung CD11b+ DCs were not changed, they were metabolically reprogrammed to skew allergic inflammation from eosinophilic Th2 to neutrophilic Th17 polarity. The mechanism for this change was independent of translational control, but dependent on inflammatory DC which produced IL-23 and increased fatty acid oxidation. mTOR therefore mediates metabolic adaptation of APCs in distinct tissues, influencing the immunological character of allergic inflammation.
Institute
Emory University
Last NameLi; Gardinassi
First NameShuzhao; Luiz
AddressEmory Woodruff Memorial Research Building, 1639 Pierce Dr NE, Atlanta, GA 30322
Emailshuzhao.li@gmail.com; luiz.gardinassi@emory.edu
Phone404-712-2988 or 404-727-5091
Submit Date2017-06-21
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2018-07-17
Release Version1
Shuzhao Li Shuzhao Li
Luiz Gardinassi Luiz Gardinassi
https://dx.doi.org/10.21228/M8BG7G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR000462
Project DOI:doi: 10.21228/M8BG7G
Project Title:Metabolomics analysis of different mouse APC subsets from lung and spleen of WT and mTOR-APC-KO
Project Summary:Antigen presenting cells from WT or mTOR-APC-KO mice were sorted from lung and spleens and subjected to metabolomics analysis.
Institute:Emory University
Department:Department of Medicine
Laboratory:Clinical Biomarkers Laboratory
Last Name:Li;Gardinassi
First Name:Shuzhao;Luiz
Address:Whitehead Biomedical Research Building, Suit 205, 615 Michael Street, Atlanta, GA 30322
Email:shuzhao.li@gmail.com;luiz.gardinassi@emory.edu
Phone:404-712-2988

Subject:

Subject ID:SU000680
Subject Type:cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:WT or mTOR-APC-KO/C57BL6
Species Group:Mammal

Factors:

Subject type: cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype HDM_stimulated
SA037736VT_150922_137mTOR-APC-KO No
SA037737VT_150922_139mTOR-APC-KO No
SA037738VT_150922_141mTOR-APC-KO No
SA037739VT_150922_165mTOR-APC-KO No
SA037740VT_150922_171mTOR-APC-KO No
SA037741VT_150922_173mTOR-APC-KO No
SA037742VT_150922_169mTOR-APC-KO No
SA037743VT_150922_167mTOR-APC-KO No
SA037744VT_150922_163mTOR-APC-KO No
SA037745VT_150922_135mTOR-APC-KO No
SA037746VT_150922_143mTOR-APC-KO No
SA037747VT_150922_105mTOR-APC-KO No
SA037748VT_150922_055mTOR-APC-KO No
SA037749VT_150922_035mTOR-APC-KO No
SA037750VT_150922_133mTOR-APC-KO No
SA037751VT_150922_059mTOR-APC-KO No
SA037752VT_150922_063mTOR-APC-KO No
SA037753VT_150922_061mTOR-APC-KO No
SA037754VT_150922_033mTOR-APC-KO No
SA037755VT_150922_031mTOR-APC-KO No
SA037756VT_150922_021mTOR-APC-KO No
SA037757VT_150922_019mTOR-APC-KO No
SA037758VT_150922_023mTOR-APC-KO No
SA037759VT_150922_025mTOR-APC-KO No
SA037760VT_150922_029mTOR-APC-KO No
SA037761VT_150922_027mTOR-APC-KO No
SA037762VT_150922_065mTOR-APC-KO No
SA037763VT_150922_057mTOR-APC-KO No
SA037764VT_150922_103mTOR-APC-KO No
SA037765VT_150922_101mTOR-APC-KO No
SA037766VT_150922_107mTOR-APC-KO No
SA037767VT_150922_067mTOR-APC-KO No
SA037768VT_150922_131mTOR-APC-KO No
SA037769VT_150922_129mTOR-APC-KO No
SA037770VT_150922_099mTOR-APC-KO No
SA037771VT_150922_127mTOR-APC-KO No
SA037772VT_150922_069mTOR-APC-KO No
SA037773VT_150922_097mTOR-APC-KO No
SA037774VT_150922_091mTOR-APC-KO No
SA037775VT_150922_071mTOR-APC-KO No
SA037776VT_150922_095mTOR-APC-KO No
SA037777VT_150922_093mTOR-APC-KO No
SA037778VT_150922_207mTOR-APC-KO YES
SA037779VT_150922_205mTOR-APC-KO YES
SA037780VT_150922_209mTOR-APC-KO YES
SA037781VT_150922_203mTOR-APC-KO YES
SA037782VT_150922_197mTOR-APC-KO YES
SA037783VT_150922_193mTOR-APC-KO YES
SA037784VT_150922_195mTOR-APC-KO YES
SA037785VT_150922_199mTOR-APC-KO YES
SA037786VT_150922_201mTOR-APC-KO YES
SA037682VT_150922_123WT No
SA037683VT_150922_125WT No
SA037684VT_150922_121WT No
SA037685VT_150922_161WT No
SA037686VT_150922_113WT No
SA037687VT_150922_115WT No
SA037688VT_150922_051WT No
SA037689VT_150922_053WT No
SA037690VT_150922_111WT No
SA037691VT_150922_117WT No
SA037692VT_150922_159WT No
SA037693VT_150922_073WT No
SA037694VT_150922_085WT No
SA037695VT_150922_087WT No
SA037696VT_150922_089WT No
SA037697VT_150922_157WT No
SA037698VT_150922_083WT No
SA037699VT_150922_081WT No
SA037700VT_150922_075WT No
SA037701VT_150922_077WT No
SA037702VT_150922_079WT No
SA037703VT_150922_049WT No
SA037704VT_150922_045WT No
SA037705VT_150922_015WT No
SA037706VT_150922_017WT No
SA037707VT_150922_109WT No
SA037708VT_150922_119WT No
SA037709VT_150922_013WT No
SA037710VT_150922_011WT No
SA037711VT_150922_003WT No
SA037712VT_150922_005WT No
SA037713VT_150922_007WT No
SA037714VT_150922_009WT No
SA037715VT_150922_001WT No
SA037716VT_150922_145WT No
SA037717VT_150922_039WT No
SA037718VT_150922_041WT No
SA037719VT_150922_043WT No
SA037720VT_150922_155WT No
SA037721VT_150922_037WT No
SA037722VT_150922_153WT No
SA037723VT_150922_147WT No
SA037724VT_150922_149WT No
SA037725VT_150922_151WT No
SA037726VT_150922_047WT No
SA037727VT_150922_175WT YES
SA037728VT_150922_187WT YES
SA037729VT_150922_189WT YES
SA037730VT_150922_191WT YES
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Collection:

Collection ID:CO000674
Collection Summary:Lungs and Spleen of WT or mTOR-APC-KO mice were aseptically removed. Spleens were finely chopped with a scalpel in 6-well plates, and digested with 1-1.5mg/mL collagenase type 4 (Worthington Biochemical Corporation) dissolved in HBSS. Digestion was performed for 30 minutes at 37°C, and reactions were stopped with addition of 2mM EDTA. Digestions were passed through 40μm cell strainers to obtain a homogenous cell suspension. To obtain lung suspensions, mice were sacrificed with CO2 and perfused with 5mL PBS injected into the right ventricle. Lungs were dissected into gentleMACS™ C tubes (Miltenyi) and dissociated using a gentleMACS™ octo dissociator using the pre-set program m_lung_01_02. Dissociated lung was digested with 1-1.5mg/mL collagenase type 4 for 30 minutes at 37°C. Lungs were next homogenized with gentleMACS™ octo dissociator program m_lung_02_01, reactions were stopped with 2mM EDTA and digestions passed through 40μm cell strainers. Cells were pelleted (1500-2000rpm/300- 500xg for 5 minutes). Red blood cells were lysed with 1mL Ack lysing buffer (Lonza) for 1 minute and lysis was stopped by addition of ≥ 5 volumes of HBSS. Cells were washed once to obtain single cell suspensions. APCs were enriched with CD11c and CD11b microbeads and MACS manual separation (Miltenyi), according to the manufacturer’s instructions. Enriched cells were stained with live/dead Aqua or Via-Probe™ (BD) and with surface antibody cocktails. Populations were further purified to ≥95% purity with a BD FACSAria cell sorter.
Sample Type:Sorted cells

Treatment:

Treatment ID:TR000694
Treatment Summary:No treatment

Sample Preparation:

Sampleprep ID:SP000687
Sampleprep Summary:Acetonitrile (2:1, v/v) was added to cell extracts, wih 3.25ul internal standard. Proteins were removed by centrifugation at 14,000g for 10 min at 4 °C. The supernatant was transferred into autosampler vials for LC-MS analysis.
Processing Method:precipitation of protein, centrifuge, and remove supernatant
Processing Storage Conditions:on ice
Extraction Method:1:2 sample:acetonitrile
Extract Storage:supernatant pipette into autosampler vials for mass spectrometer
Sample Spiking:internal standards: [13C6]-D-glucose, [15N]-indole, [15N,13C5]-L-methionine, [2-15N]-L-lysine dihydrochloride, [13C5]-L-glutamic acid, [15N]-L-tyrosine, [15N2]-uracil, [3,3-13C2]-cystine, [trimethyl-13C3]-caffeine, [U-13C5, U-15N2]-L-glutamine

Combined analysis:

Analysis ID AN001003
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Dionex Ultimate 3000
Column Thermo Accucore HILIC (100 x 2.1mm,2.6um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode POSITIVE
Units au

Chromatography:

Chromatography ID:CH000720
Chromatography Summary:Untargeted analysis with HILIC positive mode.
Methods Filename:Metab_HFQE_Setup.docx
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Thermo Accucore HILIC (100 x 2.1mm,2.6um)
Column Temperature:30
Flow Gradient:0-2min, 10%A-80%B-10%C; 2-7min, 80%A-10%B-10%C; 7-10min, 80%A-10%B-10%C.
Flow Rate:.350 ul/min
Internal Standard:internal standards: [13C6]-D-glucose, [15N]-indole, [15N,13C5]-L-methionine, [2-15N]-L-lysine dihydrochloride, [13C5]-L-glutamic acid, [15N]-L-tyrosine, [15N2]-uracil, [3,3-13C2]-cystine, [trimethyl-13C3]-caffeine, [U-13C5, U-15N2]-L-glutamine
Sample Injection:10ul
Sampling Cone:HESI probe with S-lens combination for ESI
Solvent A:100% water(A), 100% acetonitrile(B), 100% water; 2% formic acid(C)
Solvent B:100% water(A), 100% acetonitrile(B), 100% water; 2% formic acid(C)
Analytical Time:10min
Weak Wash Solvent Name:water with 10% methanol
Chromatography Type:HILIC

MS:

MS ID:MS000898
Analysis ID:AN001003
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
Capillary Temperature:300C
Capillary Voltage:44V
Ionization:electrospray ionization
Mass Accuracy:10ppm
Spray Voltage:3.5 kV
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