Summary of Study ST003047
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 PR001897. The data can be accessed directly via it's Project DOI: 10.21228/M8HH8R 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.
| Study ID | ST003047 |
| Study Title | Defective mitochondria remodelling in B cells leads to an aged immune response |
| Study Summary | The germinal centre (GC) reaction requires a unique bioenergetic supply. Although mitochondria are remodelled upon antigen stimulation, mitochondrial function in B cells is still poorly understood. To gain a better understanding of the role of mitochondria in B cell function, we generated mice that lack, specifically in B cells, Tfam, a transcription factor necessary for mitochondrial biogenesis. Tfam knock-out (KO) mice displayed a blockage of the GC reaction and established an immune response featured by the differentiation of activated B cells towards memory B cells and aged-related B cells, hallmarks of an aged immune response. Unexpectedly, GC blockage in Tfam KO mice did not cause defects in the bioenergetic supply, but this phenotype was associated with a defect in the remodelling of the lysosomal compartment in B cells. Therefore, these results may describe a new mitochondrial function for antigen presentation during the GC reaction, the abrogation of which may be the basis of an aged immune response. |
| Institute | Consejo Superior de Investigaciones Científicas |
| Last Name | Martínez |
| First Name | Nuria |
| Address | Calle Nicolás Cabrera, 1, Madrid, Madrid, 28049, Spain |
| nmartinez@cbm.csic.es | |
| Phone | 0034-911964517 |
| Submit Date | 2024-01-17 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | d |
| Analysis Type Detail | GC-MS |
| Release Date | 2024-02-05 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001897 |
| Project DOI: | doi: 10.21228/M8HH8R |
| Project Title: | Defective mitochondria remodelling in B cells leads to an aged immune response |
| Project Summary: | The germinal centre (GC) reaction requires a unique bioenergetic supply. Although mitochondria are remodelled upon antigen stimulation, mitochondrial function in B cells is still poorly understood. To gain a better understanding of the role of mitochondria in B cell function, we generated mice that lack, specifically in B cells, Tfam, a transcription factor necessary for mitochondrial biogenesis. Tfam knock-out (KO) mice displayed a blockage of the GC reaction and established an immune response featured by the differentiation of activated B cells towards memory B cells and aged-related B cells, hallmarks of an aged immune response. Unexpectedly, GC blockage in Tfam KO mice did not cause defects in the bioenergetic supply, but this phenotype was associated with a defect in the remodelling of the lysosomal compartment in B cells. Therefore, these results may describe a new mitochondrial function for antigen presentation during the GC reaction, the abrogation of which may be the basis of an aged immune response. |
| Institute: | Consejo Superior de Investigaciones Científicas |
| Last Name: | Martínez-Martín |
| First Name: | Nuria |
| Address: | Calle Nicolás Cabrera, 1, Madrid, Madrid, 28049, Spain |
| Email: | nmartinez@cbm.csic.es |
| Phone: | 0034-911964517 |
Subject:
| Subject ID: | SU003162 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA330835 | KO_F | Knock-out |
| SA330836 | KO_E | Knock-out |
| SA330837 | KO_D | Knock-out |
| SA330838 | WT_B | Wild-type |
| SA330839 | WT_C | Wild-type |
| SA330840 | WT_A | Wild-type |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO003155 |
| Collection Summary: | Tfamfl/fl mice (Larsson et al., 1998) were provided by Larsson NG (Max Plack Institute for Biology of Ageing, Cologne, Germany) and were crossed at the Centro de Biologia Molecular Severo Ochoa (CBMSO) animal facility with MB1Cre mice (Hobeika et al., 2006). MB1Cre mice were kindly provided by Alarcón B (CBMSO, Madrid, Spain). Mouse colonies were bred at the CBMSO under specific pathogen-free conditions and on C57BL/6 background. Mice were group-housed, have not been used in previous procedures and were fed with standard chow. Littermates were randomly assigned to experimental groups. Male and female between the ages of 8-16 weeks were used for the experiment. Splenic naive B lymphocytes were purified using negative B cell cell isolation kits (Miltenyi Biotec). After 24 hours stimulation, live cells were sorted, pelleted and frozen with liquid N2. |
| Collection Protocol Filename: | Protocol_BL.pdf |
| Sample Type: | B-cells |
Treatment:
| Treatment ID: | TR003171 |
| Treatment Summary: | Mice were group-housed and fed with standard chow. B lympchocytes from wild-type and knock-out mice were isolated and cultured for 24 hours in RPMI 1640 supplemented with 10% FCS, 1mM Glutamine, 0,01% sodium pyruvate, 20mM 2-mercaptoethanol, penicillin and streptomycin. Cells were cultured adding interleukine-4 and interleukine-5 at 10 ng/Ml, anti-IgM 1 ug/mL and anti-CD40 0,3 ug/mL. |
Sample Preparation:
| Sampleprep ID: | SP003168 |
| Sampleprep Summary: | From wild-type samples WT_A, WT_B and WT_C 5.6 x 106, 6.4 x 106 and 4.87 x 106 B lymphocytes were harvested respectively, and for knock-out samples KO_D, KO_E and KO_F 1.8 x 106, 2.1 x 106 and 1.8 x 106 B lymphocytes were harvested respectively. Cell homogenates were prepared by adding 1:1 v/v water: methanol and sonicated with a dr.hielscher UP200S (dr.hielscher, Berlin, Germany), set up to 80% intensity, 0.5 s/pulse, 16 pulses. For GC-MS, 100 µL of each homogenate was mixed with 300 µL of cold methanol containing 25 ppm D-palmitic acid (internal standard), vortex mixed for 60 min and centrifuged at 4000 g for 20 min. 250 µL of the supernatant were transferred to a vial with a glass insert and evaporated to dryness in a Gyrozen HyperVac VC2124 (Gimpo, Korea) coupled to a LVS 110Z (Gardner Denver Thomas GmbH Welch Vacuum, Fürstenfeldbruck, Germany). The samples were then submitted to methoximation (with O-methoxyamine) for 16 h and silylation for 1 h at 70ºC with N,O-Bis(trime5j,thylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TCMS), and finally resuspended in 100 µL heptane. |
| Sampleprep Protocol Filename: | Protocol_BL.pdf |
Combined analysis:
| Analysis ID | AN004998 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 8890 |
| Column | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| MS Type | EI |
| MS instrument type | Single quadrupole |
| MS instrument name | Agilent 5977B |
| Ion Mode | POSITIVE |
| Units | relative abundance |
Chromatography:
| Chromatography ID: | CH003776 |
| Chromatography Summary: | For GC-MS, the samples were injected onto a DB5-MS column (30 m x 0.250 mm, 0.25 µm) with a 10 m pre-column (Agilent Technologies, Santa Clara CA, USA). Helium was used as mobile phase at constant flow rate (1 mL/min) in a GC-Q-MS (8890 GC coupled to 5977B MS) system (Agilent Technologies) with an Electron Ionization (EI) source at 70 eV. |
| Methods Filename: | Protocol_BL.pdf |
| Instrument Name: | Agilent 8890 |
| Column Name: | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| Column Temperature: | -60 °C-325/350 °C |
| Flow Gradient: | NA |
| Flow Rate: | 1 mL/min |
| Solvent A: | NA |
| Solvent B: | NA |
| Chromatography Type: | GC |
MS:
| MS ID: | MS004738 |
| Analysis ID: | AN004998 |
| Instrument Name: | Agilent 5977B |
| Instrument Type: | Single quadrupole |
| MS Type: | EI |
| MS Comments: | Cell homogenates were prepared by adding 1:1 v/v water: methanol and sonicated with a dr.hielscher UP200S (dr.hielscher, Berlin, Germany), set up to 80% intensity, 0.5 s/pulse, 16 pulses. For GC-MS, 100 μL of each homogenate was mixed with 300 μL of cold methanol containing 25 ppm D-palmitic acid (internal standard), vortex mixed for 60 min and centrifuged at 4000 g for 20 min. 250 μL of the supernatant were transferred to a vial with a glass insert and evaporated to dryness in a Gyrozen HyperVac VC2124 (Gimpo, Korea) coupled to a LVS 110Z (Gardner Denver Thomas GmbH Welch Vacuum, Fürstenfeldbruck, Germany). The samples were then submitted to methoximation (with O-methoxyamine) for 16 h and silylation for 1 h at 70ºC with N,O-Bis(trime5j,thylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TCMS), and finally resuspended in 100 μL heptane. The samples were injected onto a DB5-MS column (30 m x 0.250 mm, 0.25 μm) with a 10 m pre-column (Agilent Technologies, Santa Clara CA, USA). Helium was used as mobile phase at constant flow rate (1 mL/min) in a GC-Q-MS 5975 system (Agilent Technologies) with an Electron Ionization (EI) source at 70 eV. |
| Ion Mode: | POSITIVE |
