Summary of Study ST002987

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 PR001860. The data can be accessed directly via it's Project DOI: 10.21228/M8942T 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	ST002987
Study Title	Activation induces shift in nutrient utilization that differentially impacts cell functions in human neutrophils
Study Summary	Neutrophils could utilize a variety of metabolic sources to support their vital functions as the first responders in innate immunity. Here, using in vivo and ex vivo isotopic tracing, we quantitatively examined the contributions of different nutrients under specific conditions, and found that human circulating neutrophils, in contrast to neutrophil cell line, rely on glycogen storage as a major metabolic source under resting state, but rapidly switch to primarily using extracellular glucose upon activation with various stimuli. This shift is driven by a substantial increase in glucose uptake, which is mechanistically mediated by the rapid phosphorylation and translocation of GLUT1, that dominates the simultaneous increase in gross glycogen cycling. Shift in nutrient utilization impacts neutrophil functions in a function-specific manner: Oxidative burst depends on glucose utilization; whereas NETosis and phagocytosis can be flexibly supported by either glucose or glycogen; and neutrophil migration and fungal control are enhanced by the shift from glycogen utilization to glucose utilization. This provides a quantitative and dynamic understanding of fundamental features in neutrophil metabolism, and elucidates how metabolic remodeling shapes neutrophil functions, which has broad health relevance.
Institute	Morgridge Institute for Research
Last Name	Britt
First Name	Emily
Address	330 N Orchard St Madison, WI 53715
Email	ebritt@wisc.edu
Phone	5633807709
Submit Date	2023-11-16
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2024-12-02
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001860
Project DOI:	doi: 10.21228/M8942T
Project Title:	Activation induces shift in nutrient utilization that differentially impacts cell functions in human neutrophils
Project Summary:	Neutrophils could utilize a variety of metabolic sources to support their vital functions as the first responders in innate immunity. Here, using in vivo and ex vivo isotopic tracing, we quantitatively examined the contributions of different nutrients under specific conditions, and found that human circulating neutrophils, in contrast to neutrophil cell line, rely on glycogen storage as a major metabolic source under resting state, but rapidly switch to primarily using extracellular glucose upon activation with various stimuli. This shift is driven by a substantial increase in glucose uptake, which is mechanistically mediated by the rapid phosphorylation and translocation of GLUT1, that dominates the simultaneous increase in gross glycogen cycling. Shift in nutrient utilization impacts neutrophil functions in a function-specific manner: Oxidative burst depends on glucose utilization; whereas NETosis and phagocytosis can be flexibly supported by either glucose or glycogen; and neutrophil migration and fungal control are enhanced by the shift from glycogen utilization to glucose utilization. This provides a quantitative and dynamic understanding of fundamental features in neutrophil metabolism, and elucidates how metabolic remodeling shapes neutrophil functions, which has broad health relevance.
Institute:	Morgridge Institute for Research
Last Name:	Britt
First Name:	Emily
Address:	330 N Orchard St, Madison, WI, 53715, USA
Email:	ebritt@wisc.edu
Phone:	5633807709

Subject:

Subject ID:	SU003100
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female
Species Group:	Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Time
SA325418	HI_PsA_GPi_Exp71_b	HI_PsA_GPi	30min
SA325419	HI_PsA_GPi_Exp51_b	HI_PsA_GPi	30min
SA325420	HI_PsA_GPi_Exp54_a	HI_PsA_GPi	30min
SA325421	HI_PsA_GPi_Exp71_a	HI_PsA_GPi	30min
SA325422	HI_PsA_GPi_Exp51_a	HI_PsA_GPi	30min
SA325423	HI_PsA_GPi_Exp54_b	HI_PsA_GPi	30min
SA325424	HI_PsA_Exp71_b	HI_PsA	30min
SA325425	HI_PsA_Exp51_b	HI_PsA	30min
SA325426	HI_PsA_Exp71_a	HI_PsA	30min
SA325427	HI_PsA_Exp51_a	HI_PsA	30min
SA325428	HI_PsA_Exp54_a	HI_PsA	30min
SA325429	HI_PsA_Exp54_b	HI_PsA	30min
SA325432	HL60_unstim_GPi_b	HL60_unstim_GPi	30min
SA325433	HL60_unstim_GPi_a	HL60_unstim_GPi	30min
SA325430	HL60 _unstim_b	HL60 _unstim	30min
SA325431	HL60 _unstim_a	HL60 _unstim	30min
SA325602	0min_invivoneut_Subject5	in_vivo_neutrophil	0min
SA325603	0min_invivoneut_Subject6	in_vivo_neutrophil	0min
SA325604	0min_invivoneut_Subject2	in_vivo_neutrophil	0min
SA325605	30min_invivoneut_Subject5	in_vivo_neutrophil	30min
SA325606	30min_invivoneut_Subject2	in_vivo_neutrophil	30min
SA325607	30min_invivoneut_Subject6	in_vivo_neutrophil	30min
SA325608	60min_invivoneut_Subject6	in_vivo_neutrophil	60min
SA325609	60min_invivoneut_Subject5	in_vivo_neutrophil	60min
SA325610	60min_invivoneut_Subject2	in_vivo_neutrophil	60min
SA325434	LPS_120min_Exp81_b	LPS_120min	120min
SA325435	LPS_120min_Exp83_b	LPS_120min	120min
SA325436	LPS_120min_Exp83_c	LPS_120min	120min
SA325437	LPS_120min_Exp84_a	LPS_120min	120min
SA325438	LPS_120min_Exp83_a	LPS_120min	120min
SA325439	LPS_120min_Exp84_b	LPS_120min	120min
SA325440	LPS_120min_Exp81_a	LPS_120min	120min
SA325441	LPS_120min_Exp81_c	LPS_120min	120min
SA325442	LPS_120min_Exp84_c	LPS_120min	120min
SA325443	LPS_30min_Exp81_c	LPS_30min	30min
SA325444	LPS_30min_Exp84_a	LPS_30min	30min
SA325445	LPS_30min_Exp84_b	LPS_30min	30min
SA325446	LPS_30min_Exp84_c	LPS_30min	30min
SA325447	LPS_30min_Exp83_c	LPS_30min	30min
SA325448	LPS_30min_Exp83_b	LPS_30min	30min
SA325449	LPS_30min_Exp81_b	LPS_30min	30min
SA325450	LPS_30min_Exp83_a	LPS_30min	30min
SA325451	LPS_30min_Exp81_a	LPS_30min	30min
SA325452	LPS_GPi_5_2_a	LPS_GPi	4hr
SA325453	LPS_GPi_4_26_c	LPS_GPi	4hr
SA325454	LPS_GPi_5_2_b	LPS_GPi	4hr
SA325455	LPS_GPi_4_26_b	LPS_GPi	4hr
SA325456	LPS_GPi_4_26_a	LPS_GPi	4hr
SA325457	LPS_GPi_4_21_b	LPS_GPi	4hr
SA325458	LPS_GPi_4_21_c	LPS_GPi	4hr
SA325459	LPS_GPi_4_21_a	LPS_GPi	4hr
SA325460	LPS_GPi_5_2_c	LPS_GPi	4hr
SA325461	LPS_5_2_a	LPS	4hr
SA325462	LPS_5_2_b	LPS	4hr
SA325463	LPS_4_21_a	LPS	4hr
SA325464	LPS_4_26_c	LPS	4hr
SA325465	LPS_4_26_b	LPS	4hr
SA325466	LPS_4_21_b	LPS	4hr
SA325467	LPS_4_21_c	LPS	4hr
SA325468	LPS_4_26_a	LPS	4hr
SA325469	LPS_5_2_c	LPS	4hr
SA325480	PMA_GPi_no_glc_Exp35_b	PMA_GPi_no_glc	30min
SA325481	PMA_GPi_no_glc_Exp33_a	PMA_GPi_no_glc	30min
SA325482	PMA_GPi_no_glc_Exp33_b	PMA_GPi_no_glc	30min
SA325483	PMA_GPi_no_glc_Exp29_b	PMA_GPi_no_glc	30min
SA325484	PMA_GPi_no_glc_Exp29_a	PMA_GPi_no_glc	30min
SA325485	PMA_GPi_no_glc_Exp35_a	PMA_GPi_no_glc	30min
SA325470	PMA_GPi_Exp22_a	PMA_GPi	30min
SA325471	PMA_GPi_Exp16_b	PMA_GPi	30min
SA325472	PMA_GPi_Exp35_a	PMA_GPi	30min
SA325473	PMA_GPi_Exp33_b	PMA_GPi	30min
SA325474	PMA_GPi_Exp16_a	PMA_GPi	30min
SA325475	PMA_GPi_Exp29_b	PMA_GPi	30min
SA325476	PMA_GPi_Exp22_b	PMA_GPi	30min
SA325477	PMA_GPi_Exp29_a	PMA_GPi	30min
SA325478	PMA_GPi_Exp35_b	PMA_GPi	30min
SA325479	PMA_GPi_Exp33_a	PMA_GPi	30min
SA325496	PMA_no_glc_Exp33_a	PMA_no_glc	30min
SA325497	PMA_no_glc_Exp33_b	PMA_no_glc	30min
SA325498	PMA_no_glc_Exp29_b	PMA_no_glc	30min
SA325499	PMA_no_glc_Exp35_a	PMA_no_glc	30min
SA325500	PMA_no_glc_Exp35_b	PMA_no_glc	30min
SA325501	PMA_no_glc_Exp29_a	PMA_no_glc	30min
SA325486	PMA_30min_Exp33_a	PMA	30min
SA325487	PMA_30min_Exp33_b	PMA	30min
SA325488	PMA_Exp22_b	PMA	30min
SA325489	PMA_Exp29_a	PMA	30min
SA325490	PMA_Exp22_a	PMA	30min
SA325491	PMA_Exp29_b	PMA	30min
SA325492	PMA_30min_Exp35_b	PMA	30min
SA325493	PMA_Exp16_a	PMA	30min
SA325494	PMA_Exp16_b	PMA	30min
SA325495	PMA_30min_Exp35_a	PMA	30min
SA325502	TNF_GPi_Exp22_a	TNF-A_GPi	30min
SA325503	TNF_GPi_Exp54_a	TNF-A_GPi	30min
SA325504	TNF_GPi_Exp51_b	TNF-A_GPi	30min
SA325505	TNF_GPi_Exp22_b	TNF-A_GPi	30min
SA325506	TNF_GPi_Exp51_a	TNF-A_GPi	30min
SA325507	TNF_GPi_Exp54_b	TNF-A_GPi	30min
SA325508	TNF_Exp6_b	TNF-A	30min

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Collection:

Collection ID:	CO003093
Collection Summary:	Human neutrophils were isolated from peripheral blood freshly collected from healthy donors, following the protocol approved by the University of Wisconsin Institutional Review Board (protocol no. 2019-1031-CP001). Informed consent was obtained from all participants. Neutrophils were isolated using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec, no. 130-104-434) followed by erythrocyte depletion (Miltenyi Biotec, no. 130-098-196) according to the manufacturer’s instructions. Neutrophils were spun down at 300g for 5 min, and resuspended in RPMI 1640 (VWR, no. VWRL0106-0500) supplemented with 5 mM L-glutamine (Fisher Scientific, no. BP379-100) and 0.1% Human Serum Albumin (Lee Biosolutions, no. 101-15) and kept at 37 °C in incubators under 5% CO2
Collection Protocol Filename:	Collection.pdf
Sample Type:	Neutrophil

Treatment:

Treatment ID:	TR003109
Treatment Summary:	To activate neutrophils, between 1.5 and 3 million neutrophils were aliquoted into 1.5-ml Eppendorf tubes. Cells were stimulated with the following stimuli as indicated in each experiment. PMA (Cayman Chemical, no. 10008014) was added to cell suspensions at the indicated concentrations. P. aeruginosa (PAK strain) were cultured overnight, sub-cultured until mid-exponential phase then opsonized in human serum (Sigma-Aldrich, no. H4522) for 30 min. Opsonized PsA was spun down at 10,000g for 1 min, washed and resuspended in PBS, then heat inactivated at 95 °C for 5 min before addition to neutrophil suspension at 10:1 bacterial/neutrophil cells. Zymosan A was opsonized by incubation with human serum (Sigma-Aldrich, no. H4522) at 37 °C for 30 min, washed twice and resuspended in PBS then added to neutrophils at the indicated concentration for stimulation. TNF-α (R&D systems, no. 210-TA-020) was dissolved in PBS to make a stock solution and added to neutrophils for stimulation. LPS (E. coli O111:B4, Sigma) was dissolved in PBS to make a stock solution then sonicated in a water bath for 10 min prior to addition to cell suspensions. In experiments involving glycogen phosphorylase inhibitor (GPi), 2-chloro-4,5-difluoro-N-[[[2-methoxy-5-[[(methylamino)carbonyl]amino]phenyl]amino] carbonyl]-benzamide (Cayman Chemical, no. 17578) were used, and GPi was added concurrently with stimulation unless otherwise noted at the indicated concentrations.

Treatment ID:

TR003109

Treatment Summary:

To activate neutrophils, between 1.5 and 3 million neutrophils were aliquoted into 1.5-ml Eppendorf tubes. Cells were stimulated with the following stimuli as indicated in each experiment. PMA (Cayman Chemical, no. 10008014) was added to cell suspensions at the indicated concentrations. P. aeruginosa (PAK strain) were cultured overnight, sub-cultured until mid-exponential phase then opsonized in human serum (Sigma-Aldrich, no. H4522) for 30 min. Opsonized PsA was spun down at 10,000g for 1 min, washed and resuspended in PBS, then heat inactivated at 95 °C for 5 min before addition to neutrophil suspension at 10:1 bacterial/neutrophil cells. Zymosan A was opsonized by incubation with human serum (Sigma-Aldrich, no. H4522) at 37 °C for 30 min, washed twice and resuspended in PBS then added to neutrophils at the indicated concentration for stimulation. TNF-α (R&D systems, no. 210-TA-020) was dissolved in PBS to make a stock solution and added to neutrophils for stimulation. LPS (E. coli O111:B4, Sigma) was dissolved in PBS to make a stock solution then sonicated in a water bath for 10 min prior to addition to cell suspensions. In experiments involving glycogen phosphorylase inhibitor (GPi), 2-chloro-4,5-difluoro-N-[[[2-methoxy-5-[[(methylamino)carbonyl]amino]phenyl]amino] carbonyl]-benzamide (Cayman Chemical, no. 17578) were used, and GPi was added concurrently with stimulation unless otherwise noted at the indicated concentrations.

Sample Preparation:

Sampleprep ID:	SP003106
Sampleprep Summary:	To extract intracellular metabolites, culture medium was removed and neutrophils were immediately washed with PBS. Each 2 million pelleted neutrophils were extracted with 150 µl of cold LC–MS-grade acetonitrile/methanol/water (40:40:20 v:v:v), and samples were spun at 20,627g for 5 min at 4 C to remove any insoluble debris. Samples were dried under N2 flow and resuspended in LC–MS-grade water as loading solvent.

Sampleprep ID:

SP003106

Sampleprep Summary:

To extract intracellular metabolites, culture medium was removed and neutrophils were immediately washed with PBS. Each 2 million pelleted neutrophils were extracted with 150 µl of cold LC–MS-grade acetonitrile/methanol/water (40:40:20 v:v:v), and samples were spun at 20,627g for 5 min at 4 C to remove any insoluble debris. Samples were dried under N2 flow and resuspended in LC–MS-grade water as loading solvent.

Combined analysis:

Analysis ID	AN004907
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	NEGATIVE
Units	Peak area

Chromatography:

Chromatography ID:	CH003702
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	30
Flow Gradient:	: 0 min, 5% B; 2.5 min, 5% B; 17 min, 95% B; 21 min, 95% B; 21.5 min, 5% B
Flow Rate:	0.2mL/min
Solvent A:	97:3 H2O/methanol, 10 mM TBA, 9 mM acetate, pH 8.2
Solvent B:	100% Methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004650
Analysis ID:	AN004907
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	-
Ion Mode:	NEGATIVE