Summary of Study ST002140

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 PR001355. The data can be accessed directly via it's Project DOI: 10.21228/M8K70K This work is supported by NIH grant, U2C- DK119886.

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Study IDST002140
Study TitleMitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling flux of the TCA cycle
Study SummaryThe function of mitochondrial respiration during B cell fate decisions and differentiation 55 remained equivocal. This study reveals that selection for mitochondrial fitness occurs during B 56 cell activation and is essential for subsequent plasma cell differentiation. By expressing a 57 mutated mitochondrial helicase in transitional B cells, we depleted mitochondrial DNA during 58 B cell maturation, resulting in reduced oxidative phosphorylation. Although no changes in 59 follicular B cell development were evident, germinal centers, class switch recombination to 60 IgG, plasma cell maturation and humoral immunity were diminished. Defective oxidative 61 phosphorylation led to aberrant flux of the tricarboxylic acid cycle and lowered the amount of 62 saturated phosphatidic acid. Consequently, mTOR activity and BLIMP1 induction were 63 curtailed whereas HIF1 _and glycolysis were amplified. Exogenous phosphatidic acid 64 increased mTOR activity in activated B cells. Hence, mitochondrial function is required and 65 selected for in activated B cells for the successful generation of functional plasma cells.
Institute
University of Erlangen-Nürnberg
DepartmentDivision of Molecular Immunology.Universitätsklinikum Erlangen, Nikolaus Fibinger Zentrum
LaboratoryProf. Mielenz
Last NameMielenz
First NameDirk
AddressNikolaus-Fiebiger-Zentrum, Glückstraße 6, 91054 Erlangen
Emaildirk.mielenz@fau.de
Phone++49 9131 8539105
Submit Date2022-04-06
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2022-05-02
Release Version1
Dirk Mielenz Dirk Mielenz
https://dx.doi.org/10.21228/M8K70K
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001355
Project DOI:doi: 10.21228/M8K70K
Project Title:Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling flux of the TCA cycle
Project Summary:The function of mitochondrial respiration during B cell fate decisions and differentiation 55 remained equivocal. This study reveals that selection for mitochondrial fitness occurs during B 56 cell activation and is essential for subsequent plasma cell differentiation. By expressing a 57 mutated mitochondrial helicase in transitional B cells, we depleted mitochondrial DNA during 58 B cell maturation, resulting in reduced oxidative phosphorylation. Although no changes in 59 follicular B cell development were evident, germinal centers, class switch recombination to 60 IgG, plasma cell maturation and humoral immunity were diminished. Defective oxidative 61 phosphorylation led to aberrant flux of the tricarboxylic acid cycle and lowered the amount of 62 saturated phosphatidic acid. Consequently, mTOR activity and BLIMP1 induction were 63 curtailed whereas HIF1 _and glycolysis were amplified. Exogenous phosphatidic acid 64 increased mTOR activity in activated B cells. Hence, mitochondrial function is required and 65 selected for in activated B cells for the successful generation of functional plasma cells.
Institute:University of Erlangen-Nuremberg Chair of Biochemistry
Last Name:Hofmann
First Name:Joerg
Address:Staudtstr.5, Erlangen, Bavaria, 91058, Germany
Email:joerg.hofmann@fau.de
Phone:+49 9131 85 8260

Subject:

Subject ID:SU002225
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Male and female

Factors:

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

mb_sample_id local_sample_id Genotype
SA205445B cells_DNT_GPL analysis 1-S05_DNTmutant
SA205446B cells_DNT_GPL analysis 1-S03_DNTmutant
SA2054475cDNTmutant
SA205448B cells_DNT_GPL analysis 2b-S02_DNT2mutant
SA2054496cDNTmutant
SA205450B cells_DNT_GPL analysis 2b-S04_DNT4mutant
SA205451B cells_DNT_GPL analysis 2b-S10_DNT10mutant
SA205452B cells_DNT_GPL analysis 2b-S08_DNT8mutant
SA205453B cells_DNT_GPL analysis 2b-S06_DNT6mutant
SA2054544cDNTmutant
SA205455B cells_DNT_GPL analysis 1-S01_DNTmutant
SA2054564aDNTmutant
SA2054574bDNTmutant
SA2054583aDNTmutant
SA2054593bDNTmutant
SA205460B cells_DNT_GPL analysis 2b-S05_Cre5wildtype
SA2054612bCrewildtype
SA205462B cells_DNT_GPL analysis 2b-S07_Cre7wildtype
SA2054632aCrewildtype
SA205464B cells_DNT_GPL analysis 2b-S03_Cre3wildtype
SA2054651bCrewildtype
SA205466B cells_DNT_GPL analysis 2b-S01_Cre1wildtype
SA2054671aCrewildtype
SA2054682cCrewildtype
SA205469B cells_DNT_GPL analysis 1-S02_Crewildtype
SA2054701cCrewildtype
SA205471B cells_DNT_GPL analysis 1-S06_Crewildtype
SA205472B cells_DNT_GPL analysis 1-S04_Crewildtype
SA2054733cCrewildtype
Showing results 1 to 29 of 29

Collection:

Collection ID:CO002218
Collection Summary:Both female and male mice were used in the experiments. Mice were maintained on a 12-h light/dark cycle with free access to food and water according to governmental rules. K320E-TWINKLE floxed mice (Baris et al., 2015) were crossed to CD23 CRE mice (Kwon et al., 2008) kindly provided by Meinrad Busslinger) to generate DNT animals. DNT mice used in these experiments had the genetic background DNT+/- CRE+/- and CRE control mice were DNT-/- CRE+/-. The WT animals used in this study were DNT-/- CRE-/- littermates. All mice were on the C57Bl/6 background. Isolation of primary murine cells from spleen and bone marrow Spleen was transferred into cold 2 % FCS (in PBS) and gently passed through a 70 µm cell strainer (BD) using the plunger of a 5 ml syringe (BD). Femur and tibia were flushed with cold 2 % FCS using a 27 G cannula (BD). Cell suspensions were pelleted by centrifugation at 300 x g for 5 min at 4°C. Erythrocytes were lysed in red blood cell-lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 100 µM EDTA) for 5min at room temperature. The reaction was stopped by adding cold 2% FCS before centrifugation at 300 x g for 5 min at 4°C. The final cell suspensions were kept in cold 2 % FCS after filtration through 30 µm mesh filter (Sysmex). In vitro cultivation of primary murine B cells Splenic B cells were cultured with a starting concentration of 0.5 x 106 cells/ ml in R10 medium (RPMI1640, 10 % fetal calf serum (FCS), 2 mM glutamate, 1 mM sodium pyruvate, 50 U/ml penicillin G, 50 μg/ml streptomycin, 50 μM β-mercaptoethanol) for 72 h at 37°C and 5% CO2, supplemented with 10 µg/ml LPS. For in vitro class switch recombination cells were seeded at 0.1 x 106 cells/ ml in R10 medium for 96 h, supplemented with 5 ng/ml transforming growth factor , 5 nM retinoic acid, 10 µg/ml anti-CD40 antibody, 10 µg/ml LPS, 100 U/ml IL4 and 10 ng/ml IL5. Ref.: Baris, O.R., Ederer, S., Neuhaus, J.F., von Kleist-Retzow, J.C., Wunderlich, C.M., Pal, M., WunderlichF.T., Peeva, V., Zsurka, G., Kunz, W.S., et al. (2015). Mosaic Deficiency in Mitochondrial Oxidative Metabolism Promotes Cardiac Arrhythmia during Aging. Cell Metab 21, 667–677.
Collection Protocol Filename:Bcellscoll Mielenz.pdf
Sample Type:B-cells

Treatment:

Treatment ID:TR002237
Treatment Summary:in vitro activated (LPS) B cells, which cannot replicate their mitochondrial DNA ("DNTwinkle") resulting in impaired respiratory chain activity and oxidative phosphorylation compared to Cre control B cells DTN Dominant Negative Twinkle (mitochondtail Helicase)

Sample Preparation:

Sampleprep ID:SP002231
Sampleprep Summary:Glycerophospholipid analysis Glycerophospholipids (PCH, PE, PI, PS, PG, PA) in B cells were analyzed by Nano- Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI-MS/MS) with direct infusion of the lipid extract (Shotgun Lipidomics): 14 to 45 x 106 cells were homogenized in 300 μl of Milli- Q water using the Precellys 24 Homogenisator (Peqlab, Erlangen, Germany) at 6.500 rpm for 30 sec. The protein content of the homogenate was routinely determined using bicinchoninic acid. To 100 μl of the homogenate 400 μl of Milli-Q water, 1.875ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PCH 17:0-20:4, 132 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 131 pmol PS 17:0-20:4, 62 pmol PG 17:0/20:4, 75 pmol PA 17:0/20:4 Avanti Polar Lipids) were added. Lipid extraction and Nano-ESI-MS/MS analysis were performed as previously described (Kumar et al., 2015). Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards. The calculated glycerophospolipid amounts were normalized to the protein content of the tissue homogenate. Metabolomics of phosphorylated metabolites und carbonic acids Experimental Setup I: Splenic B cells were isolated, activated with LPS and viable cells, only GFP+ for DNT, were sorted after 3 days using flow cytometry. Perchloric acid extraction and metabolic profiling was performed as previously published measured by LCMS/MS on an QTrap 3200 (Sciex) (Hofmann et al., 2011). Ref.: Kumar, V., Bouameur, J.E., Bar, J., Rice, R.H., Hornig-Do, H.T., Roop, D.R., Schwarz, N., Brodesser, 1120 S., Thiering, S., Leube, R.E., et al. (2015). A keratin scaffold regulates epidermal barrier 1121 formation, mitochondrial lipid composition, and activity. J Cell Biol 211, 1057–1075. Ref.: Hofmann, J., Bornke, F., Schmiedl, A., Kleine, T., and Sonnewald, U. (2011). Detecting functional groups of Arabidopsis mutants by metabolic profiling and evaluation of pleiotropic responses. 10Front Plant Sci 2, 82.

Combined analysis:

Analysis ID AN003501 AN003502
Analysis type MS MS
Chromatography type Ion Chromatography None (Direct infusion)
Chromatography system ThermoDionexICS3000 TriVersa NanoMate
Column ThermoDionexAS11/AG11 TriVersa NanoMate
MS Type ESI ESI
MS instrument type QTRAP QTRAP
MS instrument name ABI Sciex 3200 QTrap ABI Sciex 6500 QTrap
Ion Mode NEGATIVE POSITIVE
Units mol% pmol/10E6 cells

Chromatography:

Chromatography ID:CH002587
Chromatography Summary:The HPLC-system was controlled by the software Chromeleon VS 6.8 and DCMS-Link VS1.1 (Dionex) in combination with Analyst 1.4.1 (Applied Biosystems). Metabolites were separated on two IonPac AS11HC columns (2 × 250 mm; Dionex) protected by an AG11HC guard column (2 × 50 mm). The elution gradient was generated with water (eluent A) and 100 mm KOH (eluent B) within a total run time of 80 min at a flow rate of 0.25 mL min−1 and a column temperature of 35°C as follows: 0 min, 4%; 0 to 1 min, 4%; 1 to 6 min, 15%; 6 to 12 min, 19%; 12 to 22 min, 20%; 22 to 24 min, 23%; 24 to 27 min, 35%; 27 to 37 min, 38%; 37 to 39 min, 45%; 39 to 44 min, 100%; 44 to 71 min, 100%; 71 to 76 min, 4%; and 76 to 80 min, 4% eluent B. Ref.: Hofmann, J., Bornke, F., Schmiedl, A., Kleine, T., and Sonnewald, U. (2011). Detecting functional groups of Arabidopsis mutants by metabolic profiling and evaluation of pleiotropic responses. 10Front Plant Sci 2, 82.
Instrument Name:ThermoDionexICS3000
Column Name:ThermoDionexAS11/AG11
Chromatography Type:Ion Chromatography
  
Chromatography ID:CH002588
Chromatography Summary:Dried lipid extracts were dissolved in 300 μl of methanol. 20 μl of the lipid extract in methanol were loaded into 96-well plates and diluted with 20 μl of 20 mM ammonium acetate in methanol. Lipid infusion and ionization was conducted using Nano-ESI chips with the TriVersa NanoMate operated by the ChipSoft Software (Advion) under the following settings: sample infusion volume: 14 μl, volume of air to aspirate after sample: 1 μl, air gap before chip: enabled, aspiration delay: 0 s, prepiercing: with mandrel, spray sensing: enabled, cooling temperature: 14°C, gas pressure: 0.5 psi, ionization voltage: 1.4 kV, and vent headspace: enabled. Prewetting was done once. Ref.: Kumar, V., Bouameur, J.E., Bar, J., Rice, R.H., Hornig-Do, H.T., Roop, D.R., Schwarz, N., Brodesser, 1120 S., Thiering, S., Leube, R.E., et al. (2015). A keratin scaffold regulates epidermal barrier 1121 formation, mitochondrial lipid composition, and activity. J Cell Biol 211, 1057–1075. Ref.: Kumar, V., Bouameur, J.E., Bar, J., Rice, R.H., Hornig-Do, H.T., Roop, D.R., Schwarz, N., Brodesser, 1120 S., Thiering, S., Leube, R.E., et al. (2015). A keratin scaffold regulates epidermal barrier 1121 formation, mitochondrial lipid composition, and activity. J Cell Biol 211, 1057–1075.
Instrument Name:TriVersa NanoMate
Column Name:TriVersa NanoMate
Chromatography Type:None (Direct infusion)

MS:

MS ID:MS003261
Analysis ID:AN003501
Instrument Name:ABI Sciex 3200 QTrap
Instrument Type:QTRAP
MS Type:ESI
MS Comments:Mass spectrometric analysis was performed using the QTRAP 3200 (SCIEX) operated by Analyst 1.6.2. The electrospray ionization source parameters were −4,500 eV at 600°C, N2 gas pressures were 20 p.s.i. (curtaingas), 30 p.s.i. (gas1), and 20 p.s.i. (gas2), and collision gas was set to medium. The dwell time for ions was 75 ms, and scan time per cycle was 3.7 s.Scan ranges were from mass-to-charge ratio 87 to 606 (precursor ions) and mass-to-charge ratio 59 to 385 (product ions). Masstransitions (metabolite m/z mother ion / m/z daughter ion)recorded were as follows: UDP-glucose 565 / 323, glucose-1-phosphate 259 / 79, glucose-6-phosphate 259 / 97, 3-phosphoglycerate 185 / 97, phosphoenol pyruvate 167 / 79, citrate 191 / 87, isocitrate 191 / 111, malate 133 / 71, AMP 346/79, ADP 427/79, 2-oxoglutarate (aKG) 145 / 101, succinate 117 / 73, UDPNAG 606/385, Itaconate 129/85, Lactat 89/43, D-glucose 179/89, fumarate 115 / 71, E4P 199/97, ATP 427/79, UDP 404/79, G16BP 339/97 The contents of metabolites were calculated based on peak areas for precursor/product ion transitions relative to standards.
Ion Mode:NEGATIVE
  
MS ID:MS003262
Analysis ID:AN003502
Instrument Name:ABI Sciex 6500 QTrap
Instrument Type:QTRAP
MS Type:ESI
MS Comments:Mass spectrometric analysis was performed using the QTRAP 6500 (SCIEX) operated by Analyst 1.6.3. The following instrument dependent settings were used: curtain gas, 20 psi; CAD gas, medium; and interface heater temperature, 100°C. PC analysis was performed in the positive ion mode by scanning for precursors of m/z 184 at a collision energy of 35 eV. PE, PS, PG, PI, and PA measurements were performed in the positive ion mode by scanning for neutral losses of 141, 185, 189, 277, and 115 D at CE of 25 eV. The value for the declustering potential was 100 V (Özbalci et al. (2013) Methods Mol Biol 1033:3-20). Scanning was performed in a mass range of m/z 650–900 D and at a scan rate of 200 D/s. 61 MCA spectra were accumulated. Mass spectra were processed by the LipidView Software Version 1.2 (SCIEX) for identification and quantification of glycerophospholipids. Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards.
Ion Mode:POSITIVE
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