#METABOLOMICS WORKBENCH NurMar_20240117_071050 DATATRACK_ID:4585 STUDY_ID:ST003047 ANALYSIS_ID:AN004998 PROJECT_ID:PR001897
VERSION             	1
CREATED_ON             	January 19, 2024, 1:47 pm
#PROJECT
PR:PROJECT_TITLE                 	Defective mitochondria remodelling in B cells leads to an aged immune response
PR:PROJECT_SUMMARY               	The germinal centre (GC) reaction requires a unique bioenergetic supply.
PR:PROJECT_SUMMARY               	Although mitochondria are remodelled upon antigen stimulation, mitochondrial
PR:PROJECT_SUMMARY               	function in B cells is still poorly understood. To gain a better understanding
PR:PROJECT_SUMMARY               	of the role of mitochondria in B cell function, we generated mice that lack,
PR:PROJECT_SUMMARY               	specifically in B cells, Tfam, a transcription factor necessary for
PR:PROJECT_SUMMARY               	mitochondrial biogenesis. Tfam knock-out (KO) mice displayed a blockage of the
PR:PROJECT_SUMMARY               	GC reaction and established an immune response featured by the differentiation
PR:PROJECT_SUMMARY               	of activated B cells towards memory B cells and aged-related B cells, hallmarks
PR:PROJECT_SUMMARY               	of an aged immune response. Unexpectedly, GC blockage in Tfam KO mice did not
PR:PROJECT_SUMMARY               	cause defects in the bioenergetic supply, but this phenotype was associated with
PR:PROJECT_SUMMARY               	a defect in the remodelling of the lysosomal compartment in B cells. Therefore,
PR:PROJECT_SUMMARY               	these results may describe a new mitochondrial function for antigen presentation
PR:PROJECT_SUMMARY               	during the GC reaction, the abrogation of which may be the basis of an aged
PR:PROJECT_SUMMARY               	immune response.
PR:INSTITUTE                     	Consejo Superior de Investigaciones Científicas
PR:LAST_NAME                     	Martínez-Martín
PR:FIRST_NAME                    	Nuria
PR:ADDRESS                       	Calle Nicolás Cabrera, 1, Madrid, Madrid, 28049, Spain
PR:EMAIL                         	nmartinez@cbm.csic.es
PR:PHONE                         	0034-911964517
#STUDY
ST:STUDY_TITLE                   	Defective mitochondria remodelling in B cells leads to an aged immune response
ST:STUDY_SUMMARY                 	The germinal centre (GC) reaction requires a unique bioenergetic supply.
ST:STUDY_SUMMARY                 	Although mitochondria are remodelled upon antigen stimulation, mitochondrial
ST:STUDY_SUMMARY                 	function in B cells is still poorly understood. To gain a better understanding
ST:STUDY_SUMMARY                 	of the role of mitochondria in B cell function, we generated mice that lack,
ST:STUDY_SUMMARY                 	specifically in B cells, Tfam, a transcription factor necessary for
ST:STUDY_SUMMARY                 	mitochondrial biogenesis. Tfam knock-out (KO) mice displayed a blockage of the
ST:STUDY_SUMMARY                 	GC reaction and established an immune response featured by the differentiation
ST:STUDY_SUMMARY                 	of activated B cells towards memory B cells and aged-related B cells, hallmarks
ST:STUDY_SUMMARY                 	of an aged immune response. Unexpectedly, GC blockage in Tfam KO mice did not
ST:STUDY_SUMMARY                 	cause defects in the bioenergetic supply, but this phenotype was associated with
ST:STUDY_SUMMARY                 	a defect in the remodelling of the lysosomal compartment in B cells. Therefore,
ST:STUDY_SUMMARY                 	these results may describe a new mitochondrial function for antigen presentation
ST:STUDY_SUMMARY                 	during the GC reaction, the abrogation of which may be the basis of an aged
ST:STUDY_SUMMARY                 	immune response.
ST:INSTITUTE                     	Consejo Superior de Investigaciones Científicas
ST:LAST_NAME                     	Martínez
ST:FIRST_NAME                    	Nuria
ST:ADDRESS                       	Calle Nicolás Cabrera, 1, Madrid, Madrid, 28049, Spain
ST:EMAIL                         	nmartinez@cbm.csic.es
ST:PHONE                         	0034-911964517
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#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           	-	WT_A	Genotype:Wild-type	RAW_FILE_NAME=A_1.D A_2.D A_3.D
SUBJECT_SAMPLE_FACTORS           	-	WT_B	Genotype:Wild-type	RAW_FILE_NAME=B_1.D B_2.D B_3.D
SUBJECT_SAMPLE_FACTORS           	-	WT_C	Genotype:Wild-type	RAW_FILE_NAME=C_1.D C_2.D C_3.D
SUBJECT_SAMPLE_FACTORS           	-	KO_D	Genotype:Knock-out	RAW_FILE_NAME=D_1.D D_2.D D_3.D
SUBJECT_SAMPLE_FACTORS           	-	KO_E	Genotype:Knock-out	RAW_FILE_NAME=E_1.D E_2.D E_3.D
SUBJECT_SAMPLE_FACTORS           	-	KO_F	Genotype:Knock-out	RAW_FILE_NAME=F_1.D F_2.D F_3.D
#COLLECTION
CO:COLLECTION_SUMMARY            	Tfamfl/fl mice (Larsson et al., 1998) were provided by Larsson NG (Max Plack
CO:COLLECTION_SUMMARY            	Institute for Biology of Ageing, Cologne, Germany) and were crossed at the
CO:COLLECTION_SUMMARY            	Centro de Biologia Molecular Severo Ochoa (CBMSO) animal facility with MB1Cre
CO:COLLECTION_SUMMARY            	mice (Hobeika et al., 2006). MB1Cre mice were kindly provided by Alarcón B
CO:COLLECTION_SUMMARY            	(CBMSO, Madrid, Spain). Mouse colonies were bred at the CBMSO under specific
CO:COLLECTION_SUMMARY            	pathogen-free conditions and on C57BL/6 background. Mice were group-housed, have
CO:COLLECTION_SUMMARY            	not been used in previous procedures and were fed with standard chow.
CO:COLLECTION_SUMMARY            	Littermates were randomly assigned to experimental groups. Male and female
CO:COLLECTION_SUMMARY            	between the ages of 8-16 weeks were used for the experiment. Splenic naive B
CO:COLLECTION_SUMMARY            	lymphocytes were purified using negative B cell cell isolation kits (Miltenyi
CO:COLLECTION_SUMMARY            	Biotec). After 24 hours stimulation, live cells were sorted, pelleted and frozen
CO:COLLECTION_SUMMARY            	with liquid N2.
CO:COLLECTION_PROTOCOL_FILENAME  	Protocol_BL.pdf
CO:SAMPLE_TYPE                   	B-cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Mice were group-housed and fed with standard chow. B lympchocytes from wild-type
TR:TREATMENT_SUMMARY             	and knock-out mice were isolated and cultured for 24 hours in RPMI 1640
TR:TREATMENT_SUMMARY             	supplemented with 10% FCS, 1mM Glutamine, 0,01% sodium pyruvate, 20mM
TR:TREATMENT_SUMMARY             	2-mercaptoethanol, penicillin and streptomycin. Cells were cultured adding
TR:TREATMENT_SUMMARY             	interleukine-4 and interleukine-5 at 10 ng/Ml, anti-IgM 1 ug/mL and anti-CD40
TR:TREATMENT_SUMMARY             	0,3 ug/mL.
#SAMPLEPREP
SP: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
SP:SAMPLEPREP_SUMMARY            	lymphocytes were harvested respectively, and for knock-out samples KO_D, KO_E
SP:SAMPLEPREP_SUMMARY            	and KO_F 1.8 x 106, 2.1 x 106 and 1.8 x 106 B lymphocytes were harvested
SP:SAMPLEPREP_SUMMARY            	respectively. Cell homogenates were prepared by adding 1:1 v/v water: methanol
SP:SAMPLEPREP_SUMMARY            	and sonicated with a dr.hielscher UP200S (dr.hielscher, Berlin, Germany), set up
SP:SAMPLEPREP_SUMMARY            	to 80% intensity, 0.5 s/pulse, 16 pulses. For GC-MS, 100 µL of each homogenate
SP:SAMPLEPREP_SUMMARY            	was mixed with 300 µL of cold methanol containing 25 ppm D-palmitic acid
SP:SAMPLEPREP_SUMMARY            	(internal standard), vortex mixed for 60 min and centrifuged at 4000 g for 20
SP:SAMPLEPREP_SUMMARY            	min. 250 µL of the supernatant were transferred to a vial with a glass insert
SP:SAMPLEPREP_SUMMARY            	and evaporated to dryness in a Gyrozen HyperVac VC2124 (Gimpo, Korea) coupled to
SP:SAMPLEPREP_SUMMARY            	a LVS 110Z (Gardner Denver Thomas GmbH Welch Vacuum, Fürstenfeldbruck,
SP:SAMPLEPREP_SUMMARY            	Germany). The samples were then submitted to methoximation (with O-methoxyamine)
SP:SAMPLEPREP_SUMMARY            	for 16 h and silylation for 1 h at 70ºC with
SP:SAMPLEPREP_SUMMARY            	N,O-Bis(trime5j,thylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane
SP:SAMPLEPREP_SUMMARY            	(TCMS), and finally resuspended in 100 µL heptane.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	Protocol_BL.pdf
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	For GC-MS, the samples were injected onto a DB5-MS column (30 m x 0.250 mm, 0.25
CH:CHROMATOGRAPHY_SUMMARY        	µm) with a 10 m pre-column (Agilent Technologies, Santa Clara CA, USA). Helium
CH:CHROMATOGRAPHY_SUMMARY        	was used as mobile phase at constant flow rate (1 mL/min) in a GC-Q-MS (8890 GC
CH:CHROMATOGRAPHY_SUMMARY        	coupled to 5977B MS) system (Agilent Technologies) with an Electron Ionization
CH:CHROMATOGRAPHY_SUMMARY        	(EI) source at 70 eV.
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 8890
CH:COLUMN_NAME                   	Agilent DB5-MS (30m x 0.25mm, 0.25um)
CH:SOLVENT_A                     	NA
CH:SOLVENT_B                     	NA
CH:FLOW_GRADIENT                 	NA
CH:FLOW_RATE                     	1 mL/min
CH:COLUMN_TEMPERATURE            	-60 °C-325/350 °C
CH:METHODS_FILENAME              	Protocol_BL.pdf
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 5977B
MS:INSTRUMENT_TYPE               	GC-Q-MS
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Cell homogenates were prepared by adding 1:1 v/v water: methanol and sonicated
MS:MS_COMMENTS                   	with a dr.hielscher UP200S (dr.hielscher, Berlin, Germany), set up to 80%
MS:MS_COMMENTS                   	intensity, 0.5 s/pulse, 16 pulses. For GC-MS, 100 μL of each homogenate was
MS:MS_COMMENTS                   	mixed with 300 μL of cold methanol containing 25 ppm D-palmitic acid (internal
MS:MS_COMMENTS                   	standard), vortex mixed for 60 min and centrifuged at 4000 g for 20 min. 250 μL
MS:MS_COMMENTS                   	of the supernatant were transferred to a vial with a glass insert and evaporated
MS:MS_COMMENTS                   	to dryness in a Gyrozen HyperVac VC2124 (Gimpo, Korea) coupled to a LVS 110Z
MS:MS_COMMENTS                   	(Gardner Denver Thomas GmbH Welch Vacuum, Fürstenfeldbruck, Germany). The
MS:MS_COMMENTS                   	samples were then submitted to methoximation (with O-methoxyamine) for 16 h and
MS:MS_COMMENTS                   	silylation for 1 h at 70ºC with N,O-Bis(trime5j,thylsilyl)trifluoroacetamide
MS:MS_COMMENTS                   	(BSTFA) and trimethylchlorosilane (TCMS), and finally resuspended in 100 μL
MS:MS_COMMENTS                   	heptane. The samples were injected onto a DB5-MS column (30 m x 0.250 mm, 0.25
MS:MS_COMMENTS                   	μm) with a 10 m pre-column (Agilent Technologies, Santa Clara CA, USA). Helium
MS:MS_COMMENTS                   	was used as mobile phase at constant flow rate (1 mL/min) in a GC-Q-MS 5975
MS:MS_COMMENTS                   	system (Agilent Technologies) with an Electron Ionization (EI) source at 70 eV.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	relative abundance
MS_METABOLITE_DATA_START
Samples	WT_A	WT_B	WT_C	KO_D	KO_E	KO_F
Factors	Genotype:Wild-type	Genotype:Wild-type	Genotype:Wild-type	Genotype:Knock-out	Genotype:Knock-out	Genotype:Knock-out
Ribose 5-phosphate	805343E-11	435525E-11	628063E-12	772507E-11	838726E-11	873893E-11
Glucose	710102E-10	331188E-10	83992E-10	156801E-09	190153E-09	235136E-09
Glyceraldehyde 3-phosphate	203169E-11	144944E-11	779876E-12	185141E-11	143175E-11	237302E-11
2,3-Diphosphoglyceric acid	62077E-11	379483E-11	486343E-12	111923E-10	844039E-11	715233E-11
Phosphoglyceric acid	1367E-10	102332E-10	708295E-11	190945E-10	17259E-10	200347E-10
Pyruvic acid	305116E-10	141883E-10	163837E-10	185357E-10	194585E-10	195627E-10
Lactic acid	128273E-08	586084E-09	13097E-08	311464E-08	28993E-08	412701E-08
Citric acid	940017E-10	468624E-10	852507E-10	235353E-09	198886E-09	307229E-09
Isocitric acid	188443E-11	104812E-11	183235E-11	42721E-11	403529E-11	718433E-11
2-Oxoglutaric acid	234444E-11	279512E-11	143051E-11	396066E-11	523026E-11	385738E-11
Succinic acid	246231E-10	144457E-10	406087E-10	706514E-10	823075E-10	932506E-10
Fumaric acid	564814E-10	482227E-10	56522E-10	372171E-09	286029E-09	33345E-09
D-Malic acid	633677E-10	552381E-10	758761E-10	649881E-09	476175E-09	676646E-09
Oxaloacetic acid	76693E-12	133641E-11	815679E-12	368531E-11	300165E-11	316192E-11
Proline	216306E-09	100453E-09	110493E-09	266223E-09	160024E-09	449141E-09
Glutamic acid	97572E-09	308031E-09	312029E-09	129003E-08	120593E-08	217331E-08
Aspartic acid	232067E-08	105375E-08	907188E-09	945451E-09	717427E-09	787937E-09
Cholesterol	225783E-09	257801E-09	285164E-09	371537E-09	397277E-09	429418E-09
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Pubchem_ID	KEGG_ID
Ribose 5-phosphate	440101	C00117
Glucose	64689	C00031
Glyceraldehyde 3-phosphate	439168	C00118
2,3-Diphosphoglyceric acid	186004	C01159
Phosphoglyceric acid	-	-
Pyruvic acid	1060	C00022
Lactic acid	107689	C00186
Citric acid	311	C00158
Isocitric acid	1198	C00311
2-Oxoglutaric acid	51	C00026
Succinic acid	1738118	C00042
Fumaric acid	444972	C00122
D-Malic acid	92824	C00497
Oxaloacetic acid	970	C00036
Proline	145742	C00148
Glutamic acid	33032	C00302
Aspartic acid	5960	C00049
Cholesterol	5997	C00187
METABOLITES_END
#END