DOI: 10.5061/dryad.q83bk3jrq
title: Time course of metabolic variations in human neutrophils with PMA treatment alone or in combination with DPI: Part 1

description: Neutrophils, the most abundant leukocytes in human peripheral circulation,
 are crucial for the innate immune response. They are typically
 quiescent but rapidly activate in response to infection and
 inflammation, performing diverse functions such as oxidative burst,
 phagocytosis, and NETosis, which require significant metabolic
 adaptation. Deeper insights into such metabolic changes will help
 identify regulation of neutrophil functions in health and
 diseases. Due to their short lifespan and associated technical
 challenges, the metabolic processes of neutrophils are not
 completely understood. This study uses optical metabolic imaging
 (OMI), which entails optical redox ratio and fluorescence
 lifetime imaging microscopy of intrinsic metabolic coenzymes
 NAD(P)H and FAD to assess the metabolic state of single
 neutrophils. Primary human neutrophils were imaged in vitro under
 a variety of activation conditions and metabolic pathway
 inhibitors, while metabolic and functional changes were confirmed with
 mass spectrometry, oxidative burst, and NETosis measurements. Our
 findings show that neutrophils undergo rapid metabolic remodeling
 to a reduced redox state, followed by a shift to an
 oxidized redox state during activation. Additionally, single cell
 analysis reveals a heterogeneous metabolic response across
 neutrophils and donors to live pathogen infection
 (Pseudomonas aeruginosa and Toxoplasma gondii). Finally,
 consistent metabolic changes were validated with neutrophils in vivo using
 zebrafish larvae. This study demonstrates the potential of OMI as a
 versatile tool for studying neutrophil metabolism and underscores its use
 across different biological systems, offering insights into neutrophil
 metabolic activity and function at a single cell level. This data set is
 divided in two parts. Part 1: Time course of metabolic variations in human
 neutrophils with PMA treatment alone or in combination with DPI. Part 2:
 Metabolic variations in human neutrophils with different combinations of
 PMA treatments (2DG, 6AN, DPI, and AA)
description: For extracting intracellular metabolites, neutrophils were washed
 with PBS after removing the culture medium. Pelleted neutrophils was
 extracted using 150 μL of cold acetonitrile/methanol/water (40:40:20
 v:v:v) of liquid chromatography–mass spectrometry (LC– MS) grade (for each
 2 million cells), followed by centrifugation at 20,627g for 5 minutes at
 4°C to eliminate any insoluble residue. Samples were dried under N2 flow
 followed by resuspension in LC–MS-grade water as loading solvent. The
 soluble metabolites obtained were analyzed using a Thermo Q-Exactive mass
 spectrometer connected to a Vanquish Horizon Ultra-High Performance Liquid
 Chromatograph. Metabolites were separated on a 2.1 × 100mm, 1.7 μM Acquity
 UPLC BEH C18 Column (Waters) employing a gradient of solvent A (97:3
 H2O/methanol, 10 mM TBA, 9 mM acetate, pH 8.2) and solvent B (100%
 methanol). The gradient used was: 0 min, 5% B; 2.5 min, 5% B; 17 min, 95%
 B; 21 min, 95% B; 21.5 min, 5% B. The flow rate was maintained at 0.2 ml
 min–1. Data acquisition was performed using full scan mode. Metabolite
 identification relied on exact m/z and retention time, which were
 determined using chemical standards. Data acquisition was conducted using
 Xcalibur 4.0 software and analysis on Maven. 
description: # Time course of metabolic variations in human neutrophils with PMA
 treatment alone or in combination with DPI: Part 1
 [https://doi.org/10.5061/dryad.q83bk3jrq](https://doi.org/10.5061/dryad.q83bk3jrq) ## Description of the data and file structure Metabolomic variations in human neutrophils in different conditions: unstimulated control, PMA (100nM) treatment and PMA together with NOX2 inhibitor DPI (10μM) treatment (PMS+DPI). We also also includes stimulation with LPS (20μg/L) and TNFα (5μg/L). Metabolites were extracted at 15, 30 and 60 mins for all the conditions except for LPS and TNFα, which were extracted at 60 mins. Samples were run in duplicated (indicated by A or B in the name of the sample).  Timepoints and conditions are indicated in the name of the sample.   * Blank1.mzXML : Blank sample Replicate 1 * Blank2.mzXML: Blank sample Replicate 2 * Blank3.mzXML : Blank sample Replicate 3 * LPS_60m_A.mzXML: human neutrophils stimulated with LPS for 60 minutes replicate 1 * LPS_60m_B.mzXML :human neutrophils stimulated with LPS for 60 minutes replicate 2 * PMA_15m_A.mzXML : human neutrophils treated with PMA for 15 minutes replicate 1 * PMA_15m_B.mzXML :human neutrophils treated with PMA for 15 minutes replicate 2 * PMA_30m_A.mzXML: human neutrophils treated with PMA for 30 minutes replicate 1 * PMA_30m_B.mzXML: human neutrophils treated with PMA for 30 minutes replicate 2 * PMA_60m_A.mzXML: : human neutrophils treated with PMA for 60 minutes replicate 1 * PMA_60m_B.mzXML: : human neutrophils treated with PMA for 30 minutes replicate 2 * PMA_DPI_15m_A.mzXML : human neutrophils treated with PMA and DPI  for 15 minutes replicate 1 * PMA_DPI_15m_B.mzXML: human neutrophils treated with PMA and DPI  for 15 minutes replicate 2 * PMA_DPI_30m_A.mzXML : human neutrophils treated with PMA and DPI  for 30 minutes replicate 1 * PMA_DPI_30m_B.mzXML: human neutrophils treated with PMA and DPI  for 30 minutes replicate 2 * PMA_DPI_60m_A.mzXML: human neutrophils treated with PMA and DPI  for 60 minutes replicate 1 * PMA_DPI_60m_B.mzXML:  human neutrophils treated with PMA and DPI  for 60 minutes replicate 2 * TNFa_60m_A.mzXML: human neutrophils stimulated with TNFa for 60 minutes replicate 1 * TNFa_60m_B.mzXML: human neutrophils stimulated with TNFa for 60 minutes replicate 1 * unstim_15m_A.mzXML: human neutrophils not-treated. Control 15 min.  replicate 1 * unstim_15m_B.mzXML :human neutrophils not-treated. Control 15 min.  replicate 2 * unstim_30m_A.mzXML: human neutrophils not-treated. Control 30 min.  replicate 1 * unstim_30m_B.mzXML: human neutrophils not-treated. Control 30 min.  replicate 2 * unstim_60m_A.mzXML: human neutrophils not-treated. Control 60 min.  replicate 1 * unstim_60m_B.mzXML : human neutrophils not-treated. Control 60 min.  replicate 2 ## Code/software Data analysis was performed using the Metabolomics Analysis and Visualization Engine (MAVEN) software