Summary of Study ST002499

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 PR001272. The data can be accessed directly via it's Project DOI: 10.21228/M89402 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	ST002499
Study Title	Metabolomics analysis of stress erythroid progenitors (Part 2)
Study Summary	A time course study to assess the intracellular metabolic changes of splenic Kit+ stress erythroid progenitors
Institute	Pennsylvania State University
Department	Veterinary and Biomedical Sciences
Laboratory	Paulson Lab
Last Name	Ruan
First Name	Baiye
Address	228 AVBS Building Shortlidge Road University Park, PA 16802
Email	bur27@psu.edu
Phone	814-863-6306
Submit Date	2023-02-23
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2023-03-22
Release Version	1

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Project:

Project ID:	PR001272
Project DOI:	doi: 10.21228/M89402
Project Title:	Metabolic profiles of murine stress erythroid progenitors
Project Summary:	Inflammation alters hematopoiesis, skewing production to generate myeloid effector cells at the expense of steady state erythropoiesis. To compensate, stress erythropoiesis is induced to maintain homeostasis until the inflammation is resolved. Unlike the constant production of steady state erythropoiesis, stress erythropoiesis generates a bolus of new erythrocytes by first producing immature progenitor cells, which then transition to committed erythroid progenitors and differentiate. We hypothesize that the proliferation of early progenitor cells and their transition to differentiation is regulated by changes in metabolism. Metabolomics and isotope tracing analysis was performed to assess the intracellular metabolic profiles in proliferating progenitors isolated from in vitro stress erythropoiesis cultures. We observed an active engagement of glucose metabolism in glycolysis and anabolic biosynthesis, while the levels of TCA intermediates suggested that TCA cycle and mitochondrial respiration were blocked. Concomitantly, inducible nitric oxide synthase (iNOS) was induced in progenitor cells to increase the production of nitric oxide (NO), which was demonstrated to be crucial for proliferating progenitor metabolism. Inhibition or genetic mutation of iNOS decreased NO levels resulting in the suppression of progenitor proliferation in vitro and in vivo. As evaluated by RNA-seq, inhibition of iNOS suppressed cell proliferation-related pathways including cell cycle and nucleotide metabolism, while upregulating erythroid differentiation genes. These data suggest that iNOS-derived NO production establishes a metabolism that promotes the proliferation of progenitor cells while inhibiting their differentiation. In contrast, the transition to differentiation is marked by decreased Nos2 expression and a change in metabolism to support induction of the erythroid gene expression program. These data support a model where increased pro-inflammatory signals inhibit steady state erythropoiesis, while at the same time promoting stress erythropoiesis to maintain homeostasis.
Institute:	Pennsylvania State University
Department:	Veterinary and Biomedical Sciences
Laboratory:	Paulson Lab
Last Name:	Paulson
First Name:	Robert
Address:	228 AVBS Building, Shortlidge Road, University Park, PA 16802
Email:	rfp5@psu.edu
Phone:	814-863-6306

Subject:

Subject ID:	SU002596
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	Time_point(days)
SA250380	0-1	0
SA250381	0-5	0
SA250382	0-4	0
SA250383	0-2	0
SA250384	0-3	0
SA250385	1-5	1
SA250386	1-4	1
SA250387	1-2	1
SA250388	1-1	1
SA250389	1-3	1
SA250390	3-5	3
SA250391	3-4	3
SA250392	3-1	3
SA250393	3-3	3
SA250394	3-2	3
SA250395	8-4	8
SA250396	8-5	8
SA250397	8-3	8
SA250398	8-1	8
SA250399	8-2	8

Showing results 1 to 20 of 20

Showing results 1 to 20 of 20

Collection:

Collection ID:	CO002589
Collection Summary:	Murine spleens were harvested and disassociated to single cell suspension at a concentration of 1 x 10^8 cells/mL. Labeling reagent was added at a concentation of 50 μL/mL of sample. Cells were mixed and incubated at RT for 15 mins. Selection cocktail was added to sample at a 70 μL/mL of sample. Cells were mixed and incubated at RT for 15 mins. RapidSpheres were added to sample at a 50 μL/mL of sample. Cells were mixed and incubated at RT for 10 mins. Cells were washed with FACS buffer for 4 times, and were resuspended for downstream metabolomics analysis.
Sample Type:	Stem cells

Treatment:

Treatment ID:	TR002608
Treatment Summary:	Phenylhydrazine (PHZ)-induced hemolytic anemia To induce acute hemolytic anemia, age- and sex-matched mice were injected intraperitoneally with a single dose (100 mg/kg body weight) of freshly prepared phenylhydrazine (Sigma-Aldrich, dissolved in PBS). Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment. Brucella abortus-induced inflammatory anemia Heat-killed Brucella abortus (HKBA, strain 1119-3) was used to induce anemia of inflammation following a previously described method(1). To induce stress erythropoiesis, age- and sex-matched mice were administered with 5 x 108 particles of HKBA via intraperitoneal injection.Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment.

Treatment ID:

TR002608

Treatment Summary:

Phenylhydrazine (PHZ)-induced hemolytic anemia To induce acute hemolytic anemia, age- and sex-matched mice were injected intraperitoneally with a single dose (100 mg/kg body weight) of freshly prepared phenylhydrazine (Sigma-Aldrich, dissolved in PBS). Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment. Brucella abortus-induced inflammatory anemia Heat-killed Brucella abortus (HKBA, strain 1119-3) was used to induce anemia of inflammation following a previously described method(1). To induce stress erythropoiesis, age- and sex-matched mice were administered with 5 x 108 particles of HKBA via intraperitoneal injection.Mice were sacrificed at indicated time points for spleen sample collection and Kit cell enrichment.

Sample Preparation:

Sampleprep ID:	SP002602
Sampleprep Summary:	Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample.

Sampleprep ID:

SP002602

Sampleprep Summary:

Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample.

Combined analysis:

Analysis ID	AN004106
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000
Column	Waters XBridge C18 (50 x 2.1mm,3um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap
Ion Mode	NEGATIVE
Units	peak area normalized to IS and cell number

Chromatography:

Chromatography ID:	CH003041
Chromatography Summary:	The sample run order was randomized to reduce bias from instrument drift. 10 µl sample was subjected to LC-MS analysis on a Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Reversed-phase chromatography mode was used to separate compounds on a Xselect C18 HSS column (Waters) with solvent A (97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid ) and solvent B (methanol). The flow rate was 200 µl/min, and the total run time was 25 min. The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. The mass spectrometer was operated in a negative-ion mode at a resolution of 140,000 at m/z 200 and with a scan range of 85 to 1000 m/z.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters XBridge C18 (50 x 2.1mm,3um)
Column Temperature:	standard
Flow Gradient:	0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B.
Flow Rate:	200 µl/min
Solvent A:	97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid
Solvent B:	methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003853
Analysis ID:	AN004106
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data files were converted to .mzML file format using the ProteoWizard software, and they were analyzed by the MS-DIAL software. Metabolites were identified by comparison to an in-house reference library of pure metabolite standards which included mass-to-charge ratio (m/z) and retention time. For quantification of metabolite abundance, peak areas of identified metabolites were first normalized to the internal standard chlorpropamide, and then normalized to cell numbers from each sample. Data were analyzed using R and Cytoscape software.
Ion Mode:	NEGATIVE